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DAMI'LNG-OFF IN FOREST NURSERIES 



A THESIS ACCEPTED IN PARTIAL SATISIW TION "I' 

THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

AT THE UNIVERSITY OF CALIFORNIA 



BY 

CARL HARTLEY 



December, 1919 



<< 



UNITED STATES DEPARTMENT OF AGRICULTURE 
BULLETIN No. 934 

Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR. Chief 



Washington, D. C. 



PROFESSIONAL PAPER 



June 16, 1921 



DAMPING-OFF IN FOREST NURSERIES 



By 

CARL HARTLEY, formerly Pathologist, Office of 
Investigations in Forest Pathology 



CONTENTS 



Page 

Damping-Off in General 1 

Damping-Off of Conifers 7 

Causal Fungi 27 

Relative Importance of the Damping-Off 

Fungi on Conifers 65 

Damping-Off Fungi as Causes of Root- 
Rot and Late Damping-Off 70 

Relation of Environmental Factors to 

Damping-Off 73 



Page 

Density of Sowing 74 

Moisture and Temperature Factors ... 75 

Chemical Factors 79 

Biologic Factors 82 

Acknowledgments 86 

Summary 86 

Literature Cited 91 




WASHINGTON 

GOVERNMENT PRINTING OFFICE 

1921 



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UNITED STATES DEPARTMENT OF AGRICULTURE 

fe BULLETIN No. 934 $fjy| 




s^jtrs&ju 



Contribution from the Bureau of Plant Industry 
\VM. A. TAYLOR, Chief 






Washington, D. C. 



PROFESSIONAL PAPER 



June 16, 1921 



DAMPING-OFF IN FOREST NURSERIES. 

By Cabl II\i:tm'. . formerly Pathologist, Office of Investigations in Forest 

Pathology. 



CONTENTS. 



Page. 

Damping-off in general 1 

Damping-off of conifers 7 

Causal fungi 27 

Corticium vagum -7 

Fusarium spp , 34 

Pythium debaryanum 35 

Rheospora ngi am aphani der- 
ma tus 55 

Phytophthora spp 59 

Miscellaneous phycomj c< tes >il 

Other fungi -.-, 64 

Relative importance of the damping- 
off fvinsi on conifers 65 



Page. 
Damping-off fungi as causes of root- 
rot and late damping-off 70 

Relation of environmental factors to 

damping-off 7-". 

Density of sowing 74 

Moisture and temperature factors-- 7.~> 

Chemical factors-. 70 

Biologic factors 82 

Acknowledgments S6 

Summary ' S6 

Literature cited 91 



DAMPING-OFF IN GENERAL. 



Damping-off is the commonest English name for a symptomatic 
.rmip of diseases affecting great numbers of plant species of widely 
separated phylogenetic groups. It is commonly used for any disease 
which results in the rapid decay of young succulent seedlings or soft 
cuttings. Young shoots from underground rootstocks may also be 
damped-off before they break through the soil (Go). 1 The same term 
is even used for diseases affecting the prothallia of vascular crypto- 
gams (2). The name apparently originated in the fact that the dis- 
ease is usually m<»t prevalent under excessively moist conditions. 
In those cases in which the disease becomes serious without the pres- 
ence of unusual amounts of moisture the term is a misnomer. It is, 
however, so thoroughly established in practical use that it would be 
impossible, even if desirable, to establish any other name. 

1 The serial numbers in parentheses refer to " Literature cited," at the end of this 
bulletin. 

19651°— Bull. 934—2] 1 



2 BULLETIN 934, CJ. S. DEPARTMENT OF AGRICULTURE. 

While the parasites reported as causing damping-off are probably 
not as numerous as the host species which are subject to it. a con- 
siderable number are known. Two quite different types of damping- 
off parasites may be recognized. In the first type we have fungi, 
such as Pythium debaryanum Hesse and Corticium vagum T>. and ('.. 
soil inhabiting and primarily saprophytic, which attack a great 
variety of hosts, and are at least better known, if not more destruc- 
tive, as damping-off organisms than as parasites on older plants. 
They are specialized as to the type and age of tissues which they at- 
tack rather than as to host. The second type includes fungi Less 
common as saprophytes and with a relatively limited, sometimes very 
closely limited, host range. Phoma betae, the systemic parasite of 
sugar beet (37). is an excellent example of the host-specialized para- 
site, transmitted in the seed and capable of seriously injuring various 
parts of the older plant at different stages of growth as well as at- 
tacking seedlings. 

Most damping-off parasites are intermediate in habit between the 
extremes of these two types. Of those which are somewhat host 
specialized, the following may be mentioned: 

Phomopsis reran*, the cause of foot-rot of eggplant, reported by Sherbakoff 

(128) as a frequent cause of damping-off of this host ami believed to ho 

carried on seed. 
Cribberclla sa-ubinetii (Mont.) Sacc. (29) and the imperfect fungi which kill 

grain seedlings as well as cause diseases of the older plants (80; 126, 

p. IMS). Species of Gloeosporium and Volutella named by Atkinson I 2 

p. 269; 52) as able to kill seedlings or cuttings of particular host plants. 
Glomerella (Colletotrichum) gossypii, described by Atkinson (1) and Barre 

(4) as likely to cause damping-off of cotton (112). 
Fusarium lint, the Hax parasite, reported by Bolley (14) as destructive .to 

young seedlings. 
Phoma lingam, the cause of black-leg of cabbage, at least under inoculation 

conditions able to kill quickly seedlings of cabbage and other crucifers 

(72). 
Peranospora parasitica (Pers.) De Bary, a downy mildew attacking cabbage 

and various other crucifers. reported as killing thousands of very young 

cabbage plants in Florida seed heds (41). 
The entomophthoraceous Completoria complens, on fern prothallia i 1 ; 87, 

p. 203). 
Bacillus malvacearum, a parasite of the leaves of cotton plants, which can 

also cause damping-off of its favorite host (113) and the bacteria from 

diseased cucumber plants with which Halsted (53) caused typical 

damping-off of cucumbers. 

Damping-off fungi with wider host ranges include PhytopMTiora 
fagi, Aphanomyces levis (100), Rheosporangiwm aphanidermatus 
(38, 39), Botrytis cinerea, and certain Fusaria. The so-called prop- 
agation fitngiis. " vermehrungspilz," a sterile damping-off mycelium 
which Sorauer (133, p. 321) believed related to Sclerotinia and for 
which Ruhland (115) has erected a new genus, considered by both 



i ' l 3 



DAMPING-OFF IH FOREST NUESERIES. 6 

authors the most serious enemy encountered in growing softwood 
cuttings in Germany, if distinct would be a further addition to these 
generalized parasites. However, it is now believed (H4) to be identi- 
cal with Cart /(in in vagum. Common generalized parasites of older 
plants, such as Sclerotima libertiana, Sclerotium rolfsii (129), and 
Thielavia basicola (47), capable of attacking roots or other pails of 
older plants of numerous species, may also be considered among the 
damping-off fungi when they cause the death of small seedlings, as 
occurs, for example, in attacks by Sclerotinia libertiana on lettuce 
(20, p. 28) and celery (Id-"., p. 536) in seed beds. Further study will 
probably result in multiplying almost indefinitely the number of 
more or less important damping-off parasites, both of the specialized 
and uftspecialized groups, although the most important of the latter 
type are probably already known. 

Most of the references in literature to damping-off describe its 
occurrence in truck crops and the losses caused in these crops. Ac- 
cording to Halsted (53, p. 342), weed seedlings are also very com- 
monly attacked. Duggar (33) names lettuce, celery, cotton, sugar 
beet, cress, cucumber, and sunflower as especially susceptible to 
injury by the two most important damping-off organisms. Except 
for the plant species in which damping-off by seed-carried parasites 
is common, it appears that the economic damage from damping-off 
is serious only with plants whose culture involves the raising of the 
seedlings in crowded seed beds for subsequent transplanting. For 
example, tomatoes do not ordinarily suffer from damping-off in the 
field (TO), but the growing of seedlings in flats for subsequent trans- 
planting is sometimes seriously hampered as a result of the preva- 
lence of damping-off. This same principle holds in general for trees. 
Broad-leaved trees, which are usually not as crowded in the seedling 
stage as are the conifers, seldom give rise to complaint on the score 
of damping-off. The conifers, subject to serious losses in nursery 
beds, are not believed to be greatly affected in this country by the 
better known types of damping-off under forest conditions (68) 
except in the less common cases in which seedlings come up in close 
groups from squirrel hoards, artificial seed spots, or similar sources. 
A considerable number of broad-leaved trees have been reported at 
one time or another as injured by damping-off, though complaints 
of commercially serious losses are not common. The cases which 
have come to the writer's attention are listed below: 
Cause not determined : 

Orange (43, 108). 

Olive, in greenhouse at the University of California. 

Russian wild olive (Elaeagnus sp. ), serious at an Iowa nursery; oral re- 
port by Mr. C. R. Bechtle, formerly of the United Slates Foresl Service; 
at another nursery in the same region this plant was reported as very 
little subject to injury. 

,.■ .. o, , I 



4 BULLETIN 934, U. S. DEPARTMENT or AGRICULTURE. 

Cause not determined — Continued. 

Magnolia (31), troublesome if the pulp is not washed off the seed before 
planting. 

Eucalyptus spp. (88, i». 45; L31), serious under moist conditions. 

Bet uia spp. Communication by Dr. Perley Spaulding, of the Bureau of 
riant Industry; found especially susceptible in a Pennsylvania nursery. 

Carob, at United States Plant Introduction Garden, Chico; Calif. I>r. 
.Mel T. Cook states that damping-off is more serious in carob seedlings 
if the seed is removed from the pod than if pods and seeds are sown 
together. 

Robinia pseudacacia (13). 

Apple, in greenhouse at the Michigan Agricultural College. 
Sclerotinia sp. (Europe) : 

Betula (79), a disease of seed and germinating seedlings. 
Phytophthora fagi (Europe) : 

Fagus. Elartig (59) and many other writers; seriouslj affected, even 
in forest. 

Platanus (15). 

Acer (15), l. platanoides and .1. pseudoplatanus (SO, 104). 

Robinia ("»!•. 73). 

Fraxinus (73). 

Acacia (59). 
Cercospora acerina (Europe) : 

leer platanoides and A. pseudoplatanus (58). 
Pythium debaryanum: 

Tilia europea and 'I', ulmifolia (137). serious. 

Robinia (7."), p. 13-14), killing germinating seed. 

Catalpa (126). 
Rhizoctonia : 

Citrus seed beds (130) ; much loss. 

Catalpa (126). 
Botrytis cinerea: 

Catalpa (126). 
Fusariuvi sp. : 

Citrus seed beds (130) ; much loss. 

The sugar beet is apparently the only plant whose damping-off 
diseases have been investigated with any decree of eompleteness 
by modern methods. While there is a great mass of literature on 
damping-off, it is mainly descriptive and on control measures. Most 
of the reports of the causal relation between the different fungi and 
the disease in the various host plants have been based on demon- 
strations of the presence of the fungus in diseased seedlings. In 
a great number of these cases identification has been doubtful. 
Even when a fungus is known to belong to a parasitic species, it 
is by no means certain that the mycelium found belongs to a para- 
sitic strain. It has been found, for example, that only part of 
the strains of Corticium vagum occurring in sugar beets are able 
to attack that host vigorously (38, p. 154). Similar data for pine 
appear in figures 1 and 2. Furthermore, even parasitic strains of 
several of the damping-off organisms are so widely distributed as 



DAMPING-OFF IX FOREST NURSERIES. 5 

saprophytes that one of them might easily get into a killed seedling 
after sonic other parasite had caused its death. Not only in the 
case of seedlings killed by fungi like Peronospora parasitica, but in 



HOST 
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Fig. 1. — Diagram showing the relative activity of different strains of Corticium vagum 
in inoculations made at the time of sowing the seed. In experiments Nos. 36, 45, and 
47 the values are plotted for the number of seedlings appearing above the soil. For the 
other experiments the number of seedlings surviving at the close of the experiment have 
been taken. Explanation of symbols : 0=Strain 147, from spruce seedlings, Washington, 
D. C, 1910; +=strain 50, from pine seedlings, Nebraska, 1900; D=strain 233, from 
Elaeagnus sp., Kansas, 1913; H=strain 230. from the same lesion as strain 233; 
• =strain 1S3, from bean, New York. 1910. 



HOST 
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Fio. 2. — Diagram showing the relative activity of different strains of Corticium vagum, 
as indicated by the number of seedlings surviving in inoculated soil. Explanation of 
symbols: ©=Strain 1S9, from sugar beet, Michigan, 1910 (light-brown mycelium with 
few sclerotia) ; A=strain 211 and A=strain 212, from sugar beet, Colorado, 1910; 
B=strain 186, from potato, Ohio, 1910; D=strain 1S7, from potato. New York, 1910; 
+=strain 205, from Douglas fir, Colorado, 1911; X=straiu 192 and 0=«train 206, 
from pine, Nebraska, 1911. 

cases of true damping-off produced by the rotting type of parasite, 
much of the rapid decay of the seedling after death is brought 
about by bacteria and fungi other than the one causing death. 



BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

Iii()cii]:ition experiments are therefore probably even more neces 
in \ in damping-off investigations than in studies of most other dis- 
eases in order to demonstrate etiological relationships. Unfortu 
Qately, most of the inoculation work with damping-off organisms 
prior to L900 was either crudely done by placing diseased seedlings 
against healthy ones or consisted of experiments in which purity 
of cultures and validity of controls did not receive sufficient eon 
sideration. Recent investigations not primarily directed toward 
damping-off, but which have decidedly increased our knowledge of 
the relation between Corticium and the disease, are those of Peltier 
(98) and Fred (43). The latter established a strong presumption 
that the difficulty in securing stands of various field crops having 
oily seeds in soil where green manures had been recently turned 
under is due to the killing of the sprouting seed by damping-off 
organisms. 

In tobacco, sugar beet, and pine, whose damping-off has received 
considerable attention, it has been found that the damping-off proper 
is commonly preceded by the killing of many of the sprouting seeds 
in the soil (38; 68, p. 522; 81, p. 5) and followed, after the plants 
become too large to be killed by the damping-off organisms, by root 
sickness and the death of small roots (38, p. 161 ; 64; 100). This latter 
has been reported also as a serious matter in the case of Corticw/m 
vagum for potato (51), a host on which damping-off is not important 
because of the lack of commercial propagation from seed. PytTwam 
debaryanurn further has been reported as continuing to work in the 
cortical tissues and leaves of tobacco plants which have been in- 
fected too late to result in death (81). 

The fact that a number of the damping-off fungi are able to attack 
young or soft tissues of so great a variety of plants and are much 
less able to kill older plants suggests that resistance to damping-off 
may be in part based on purely mechanical factors. Hawkins and 
Harvey (71) recently have extended to Pythium debaryanurn the 
idea, developed by Blackman and Welsford (12) and Brown (16) for 
Botrytis cinerea, of the importance of mechanical penetration in the 
fungous invasion of plant tissues. While for B. cinerea mechanical 
pressure was found to be the main factor only in cuticle penetration. 
with P. debaryanurn the penetration of the cell walls of all parts of 
the potato tuber was apparently largely dependent on mechanical 
puncturing by the hyphae, only tubers with mechanically weak cell 
walls being susceptible to decay by the fungus. The extreme sus- 
ceptibility to /'. debaryanurn and Corticium vagum of soft, thin- 
walled tissues and the resistance of older stems and root parts would 
fit in well with such a theory as to the method of wall penetration, 
as in the older tissues the thicker cell walls would obviously be a 
serious bar to the extension of a fungus dependent partly or en- 



DAMPING-OFF IX FOREST NURSERIES. 7 

tirely on mechanical puncturing Bor its progress from cell to cell. 
Hartig (61, p. 1 17 L50) shows a fungus which he does no! name, luit 
which is evidently a species of Fusarium, dissolving the young un- 

cuticularized epidermis of pine seedlings; but he stall's that it can 
not so dissolve older epidermis. The increased protective value of 
the epidermis of older plants can only in part explain the immunity 
most of them develop against serious attack by damping-off organ- 
isms, as lesions already started or which may later develop from the 
infection of young roots are unable to extend into the older pa its 
of the plants. 

It may be mentioned here that the writer in a very preliminary 
test found strains of Corticiwn vagwm and Fusa/riwm mondliforme 
Sheldon which had been proved able to cause damping-off of pines 
also apparently able to destroy filter paper in inorganic salt solu- 
tion, while Pythium debaryanum seemed not so able. Ruhland 
(116), on the other hand, found the strain of the " vermehrungspilz " 
{Corfu-/ inn vagum) which he tested to be very weak in cellulose- 
destroying ability as compared with Botrytis cinerea. 

DAMPING-OFF OF CONIFERS. 

HISTORICAL. 

While the losses from damping-off in seed beds of dicotyledonous 
tree species are occasionally serious and in the case of beech in 
Europe have required considerable study, they have been so far 
overshadowed in this country by the losses in coniferous seed beds 
that practically all the attention thus far, both as to etiology and 
measures of prevention, has been devoted to the disease in conifers. 

The literature on the damping-off of conifers is considerable. 
A large part of it, because of the extensive early development of 
plant pathology and forest planting in Germany, has been writ- 
ten by Germans. A large portion of the German articles on it 
was either by foresters or by botanists in the day when most patho- 
logical work was of the reconnaissance type. Therefore, while the 
work of one of the best known of the parasites on coniferous seed- 
lings was noticed in Europe as early as the eighteenth century (21, 
p. 252-253) most of the European data available are observational. 
The only fungi which were at all definitely connected with the dis- 
ease on conifers seem to have been Fusarium {Fuxoma spp.) and 
Phytophthora fagi (P. omnivora De Bary in part). The damping- 
off Rhizoctonia was described in Germany in L858 and Pythium de- 
baryanum in L874; the fact that neither of these, important in conif- 
erous seed beds in both the eastern and western United States, has 
ever been reported from conifers in Europe is perhaps the best evi- 



8 BULLETIN 934, V. S. DEPARTMENT OF AGRICULTURE. 

dence of the relatively small amount of actual investigation carried 
on there on this disease in the nurseries. A aumber of references to 
the damping-off of conifers in the English horticultural and botani- 
cal Literature yield even less definite information as to the causal 
fungi than do the German articles. 
• With the awakening of interest in reforestation in the United 
States between L5 and 20 years ago and the first efforts to grow 
pines in quantity for forestry purposes, attempts were made to de- 
termine the cause of the disease in this country and to develop direct- 
control methods. Duggar and Stewart (32) made what appears to 
be the first report of Rhizoctonia in- connection with the damping-off 
of conifers. Spaulding (136, 137), in work begun in L905, con- 
tributed much to our knowledge of the etiology of the damping-oil' 
of pine in this country, especially in relation to Fusarium, and origi- 
nated the sulphuric-acid method of control. The writer in L910 re- 
ported preliminary inoculations on conifers with both Rhizoctonia 
and Pythiwn debaryanum (62). The work of Gifford (46) and 
Hofmann (77) added to the information on the causal relation of 
Fusarium spp. and /'. debaryanum, respectively. Hartley. Merrill, 
and Rhoads (68) have recently established the parasitism of a num- 
ber of strains of the Corticium vagum type of Rhizoctonia on pine 
seedlings under inoculation conditions, have confirmed Spaulding's 
conclusions as to the parasitism of Fusarium moniliforme Sheldon, 
and have given preliminary data on other fungi. They consider /'. 
debaryanum and ('. vagum more important in pine seed bedsthan any 
single Fusarium species. Hartley and Halm (G9) have announced 
successful inoculations on pines with- P. debaryanum and Rheospo- 
rangium aphanidermatus Edson, with less satisfactory evidence of 
the parasitism of PhytopJithora sp. and a fungus tentatively referred 
to Pythium artotrogus. Hartley and Pierce (67) report the finding 
of P. debaryanum in Tsuga rrn rtensiana and Pseudotsuga taxifolia 
as well as in the pines. In damped-off pine seedlings they find P. 
debaryanum more commonly than ('. vagum, especially in beds which 
have received disinfectant treatments. Other considerations, how 
ever, keep them from concluding that the former is necessarily the 
more important of the two. Both of these latter papers and all of 
the reports of Pythium with the exception of Hofmann's are brief 
notes, presenting no evidence in support of the statements made. 

DESCRIPTION. 

The symptoms of damping-off in conifers have already been de- 
scribed in some detail (68). In the paper cited, injury due to exces 
sive heat of the surface soil and injury caused by high wind, both of 
which may easily be confused with damping-off, are described and 
accompanied by colored illustrations both of different types of damp- 



DAMPING-OFF in kokkst ni'kskimks. 9 

ing-off and of these nonparasitic troubles. The detailed descriptions 
will not be repeated here. A brief summary of the different types 
of disease recognized as included in damping-off follows: 

(1) Germination loss: The radicles arc killed very soon after the seeds 
sprout and before the seedlings can appear above ground. This is an important 
type, which can be caused probably by any of the organisms commonly capable 
of causing the better known types of trouble (61, 63, <»S, 137). 

(2) Normal damping-off (figs. •".. 1, and 5) : The seedlings are killed by fungi 
invading either the root or hypocotyl after the seedling has appeared above the 
soil and while the stem is still dependent largely on the turgor of its cortical tis- 
sues for support. In sandy soils root infection is more common than hypocotyl 
infection, though the latter is the type most emphasized in the early horticul- 
tural descriptions. Ruttner (26) some time ago recognized the frequence of 




Fig. 3. — Normal type of damping-off of Plnus ponderosa. At the left is a dampeil-off 
seedling or root sprout of the southwestern ragweed (Ambrosia psilostachya) . (Photo- 
graphed by S. C. Brunei-.) 

root infections. Damping-off in beds out of doors is primarily in most cases a 
root rot, either of this type or of the types preceding and following. 

(3) Late damping-off includes cases of the root-rot type occurring only after 
the seedling stems have started to become woody and the cortex has begun to 
shrivel. The damping-off parasites, or at least part of them, continue to kill 
seedlings by rotting their roots for some time after the stems become too woody 
to be decayed. The seedlings affected do not fall over till a considerable time 
after death. For convenience, all cases of this sort up to the purely arbitrary 
a.ue of two months are classed as damping-off. However, in weather permitting 
of average speed of development the seedlings are usually able to resist attack 
before they reach this age. Seedlings at the marginal age between suscepti- 
bility and nonsusceptibility to killing infections are found often with the 
younger parts of their roots killed, but with the older portions apparently able 
to resist invasion by the fungus, recovery taking place by laterals. Dr. 
R. D. Rands and the writer in 1911 established the ability of seedlings from 
4:;-day-old beds of Pin/us sylvestris, P: banksiana, P. vigra amtriaca, and P. 
nigra poiretiana to survive such infections, even when more than half of the 
root system lias been destroyed, by transplanting such root-sick seedlings and 



10 



BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 



observing their continued growth (fig. 6). An article recently found (2.""0 shows 
thai Biittner had earlier made the same sorl of demonstration of recovery of 
root-sick conifers. Observations on olive seedlings in 1916 showed cases of 
partially rotted roots which were recovering by sending out lateral root 
branches. 

(4) To]> damping: Tin- cotyledons or upper part of the stem are invaded by 
tiu 1 parasite, sometimes before the seedling breaks through the soil. The infec- 
tion may or may not be fatal. A special case of this type, probably caused by a 
different parasite from those most commonly active, is that which in a publica- 
tion above referred to was described and figured as " blacktop " (68). Tt is 




■A*- 



~* 'i^ftg*' 






(&4gMt& 









&*. 




Fiq. 4. — The ttosdiining of an epidemic in drill-sown Pinus banksiana. Black crosses (X^ 
indicate disease foci where the germinating seed were apparently killed and from which 
the disease is now spreading to adjacent seedlings. (Photographed by It. J. V. 
Hofmann.) 

distinguished from ordinary top damping by the very dark color of the invaded 
tissues and its apparent dependence on some unusual set of climatic factors for 
its progress in the seedling after infection. 

The killing- of dormant seed by fungi is a matter of some practical 
interest in seed beds, and possibly still more so in forests, as it may 
help to explain the failure of certain conifers to reproduce except on 
mineral or certain other special soil types (68). With sugar beets 
Pythium debaryanum (100) is said to attack dormant seed as well 
as seeds which have sprouted. It is to be presumed that with conifers 
some of the damping-off fungi will be found to attack dormant as 
well as sprouting seed. This matter is now under investigation. 



TUMPING-OFF IN FOREST NURSERIES. 



11 



Something is already known aboul the seed Fungi of herbaceous 
plants ("(!. 91), broad-leaved trees (7!». 92"), and juniper (95). 

RELATIVE IMPORTANCE OF THE DIFFERENT TYPES. 

Of the types of damping-off described in the foregoing pages the 

first two are ordinarily the most important. Late damping-off is 
rarely as serious as the normal type of damping-off. Top damping is 
only of importance in eases of excessive and unusual atmospheric 
moisture, so far as the writer's experience indicates. In the Middle 
West it has proved relatively insignificant. The three types which 




Fig. 



. — Nearly complete destruction of the seedlings of Pinua banksiona at an unusually 

early aye, at Garden City. Kans. (Photographed by Dr. J. V. Hofmann.) 



occur after the seedlings appear above the soil surface can, of course, 
be evaluated by frequent counts during the damping-off season. This 
has apparently not yet been done by anyone. However, in experi- 
ments on damping-off control by soil disinfection, data have been 
obtained on comparative emergence (number of seedlings appearing 
above the soil surface) in treated and untreated plats and on the total 
parasitic losses after the seedlings appear which permit a certain 
amount of analysis of the losses due to damping-off parasites. The 
data from five nurseries bearing on (his point are presented in 
Table I. 



12 



BULLETIN £>34, V. s. DEPARTMENT OF AGRICULTURE. 



Table I. -Relative importance of losses by damping-off before and after conifer 
seedlings emerge (rum the soil. 





Series. 


I; i 




I oss in control plal s. 


Nursery and species. 


Disinfectant. 


Number of 

plats. 


Emergi <i 
( [abli seeds 


Ratio 

of 
col. 6 

to 
CO] 7 




Treat- 
ed. 


Con- 
trols. 


Be- 
fore. 


afti r. 


Total. 


' 


2 


3 


4 


5 


6 


7 


8 


'.» 


Bessey i Nebraska sand hills): 

Pinus banksiaha 

.— Pinus ponderosa 


Average of 2 


Sulphuric acid 

...do 

do 


6 

t 

4 

3 
17 

6 

li 
4 
1 
I 

' (i 
2 

7 
8 


I'M 
8 
7 

3 

2 
29 

3 
3 

1 

:{ 
2 

3 

7 

8 
6 


P.ct. 
37.8 

28. I 
29.4 

80.7 
15.3 

3.8 

25. 7 
5.7 
7. 1 
1.9 

.-.. 9 
58.2 

12.6 

1 1. 5 


P.ct. 
e27.2 
27.3 
54. 1 

27.5 

12. ii 
30.9 

37 2 
36. 2 
26. -2 

13. 6 
16. 9 

15.3 
18.0 

36. 1 

18.6 


P.ct. 

65. (i 
55. I 

92. 7 
16. '-' 

ll.ii 
61. 9 
31.9 

5. 1 
21.8 

51.2 
76. 2 

18.7 

33.1 


1.03 
.54 


afclcn Citj (southwestern 
" Pinus austriaca 


Average of 2 


Copper sul- 
phate. 
Zinc chlorid.. . 

Copper sul- 
phate. 

Formaldehyde 

do :... 

Zinc chlorid. . . 
do 


2. 52 
6 72 


Pinus ponderosa 


A\ i rage of 7 

No 1051 

No. 1052 

No. 1053....1... 
No. 1054 


fi 


i 'ass Lake i northern Minni - 
sota): 


.10 

.71 
.22 




. is 




Nos. Ki57 and 
1061. 

(1073 

\1074 


Eeal . 


. 29 


Bast Tawas i Michigan i: 
I'inus resinosa 


Foi maiden] de 

Sulphuric acid 

Formaldehyde 
Sulphuric acid 


.13 




:s. 25 


Fort Bayard i New Mexico): 
i'inus ponderosa 


fNos. 791 and 

1 792. 

INos. 891 and 

I 892. 


.35 
.78 



a Area counted. 122 square feet. '< Irea counted, 78 square feel 

<■ Ii> Pinus banksiana at the Bessey Nursery, the loss after emergeni e is slightly low and the ratio slighth 
high, because of the closing of count's on a few of the series before damping-off was entirely over. 

The procedure was to average the number of seedlings which 
emerged in the control plats in each series and subtract this number 
from the average number emerging (that is. appearing above the 
soil surface) in the treated plats in the same series. The treated 
plats chosen were the ones which allowed the averaging of the 
greatest number of plats treated with the same disinfectant. Only 
those plats were taken in which there was no evidence of injury to 
the seed or seedlings by the disinfectant and in which the amount 
of normal damping-off during the first few days after emergence 
was so slight as to indicate satisfactory initial control of the parasites 
by the treatment. In such plats it was assumed that the germination 
loss was unimportant, and the average number of seedlings appear- 
ing on them was taken as representing the number of viable seeds per 
plat. The difference between this emergence figure and the average 
emergence in the controls was taken as indicating the extent of para- 
sitic loss before the seedlings appeared, including any destruction 
of dormant seed by parasites which may have occurred as well as 
the killing of germinating seed. Both this figure and the number 



DAMPlXti-Ol F IX FOREST NURSERIES. 



13 



of seedlings which succumbed to damping-off after emergence were 
reduced to a percentage based on the indicated number of viable sec. Is. 
and they are directly compared in columns 6 and 7 of Table I. At 
three of the nurseries the data of the same species of pine and with 
the same treatment were averaged. 

The data in Table 1 do not indicate any regularity either in the 
extent of loss before emergence, the loss after emergence, or in the 
ratio between these 
two values. For ob- 
vious reasons, no reg- 
ularity is to be ex- 
pected in any of these 
items. The table is 
of some interest, 
however, in confirm- 
ing the evidence of 
the inoculation ex- 
periments, of obser- 
vation of sprouting 
seed dug up in the 
beds, and of the par- 
tial or complete fail- 
ure of emergence at 
the centers of large 
damping-off foci 
(figs. 4, 7, and 8) that 
the work of parasites 
before the seedlings 
appear may in some 
cases be of consider- 
able importance. It 
is obviously impos- 
sible to make any 
general quantitative 
statement of the se- 
riousness of such loss, 
in view of the varia- 
tion in its extent at 
different times and 
places and of the in- 
accuracy of any computations based on the relative emergence 
of hosts as irregular in their germination as the conifers are 
known to be. The case is complicated in addition by the fact 
that, despite careful avoidance of treated plats known to have 
suffered chemical injury, it is probable that a few seedlings were 
killed before emergence by the disinfectants used in some of the 




Fig. ti. — Root sickness in Pinus nigra poiretiana. The two 
seedlings at the right are healthy. The three at the lefl 
have had their taproots decayed to within li inches 
of the soil surface. All are putting out lateral roots from 
the lowermost sound point. Similarly injured seedlings 
when transplanted lived and made satisfactory growth. 



14 



BULLETIN !»::t. r. S. DEPARTMENT of agriculture. 



cases. It may furthermore be that in other cases the disinfectant had 
a stimulating effect, resulting in better germination in the treated 
plats, entirely aside from that resulting from parasite control. The 
number of disinfectant methods which concurred in giving apparent 
increases in germination, however, makes it seem reasonably cer- 
tain that no great part of the increase was d\w to this stimulation. 
In addition to the different disinfectants shown in the table, mer- 
curic chlorid, heat, hydrochloric acid, nitric acid, and ammonia all 
apparently resulted in approximately the same increases of germina- 
tion in tests at the Bessey Nursery as the sulphuric acid which was 
used as the standard for comparison in most of the series. Relative 
emergence in treated and untreated plats, as well as damping-off 




Fig. 7. — A clean-killed area in a bed of Pinus ponderosa, caused by Oorticium vagum. 
Inside a 12-inch circle at the center of this "patch" no seedlings appeared. It will 
be noted that the weeds as well as the pines have been killed with the exception of 
Salsola tragus. 

loss after the seedlings appeared, was determined at two nurseries 
in addition to those given in the table. The results at these nurseries 
in general confirmed those at the five nurseries covered by the 
table in showing lower emergence in the controls. Although it is 
impossible to draw positive conclusions, some idea of the seriousness 
of losses before the appearance of the seedlings above ground can be 
obtained by studying the data in Table I. The fact that such losses 
appear considerable, sometimes exceeding the losses from the damp- 
ing-off that occurs after emergence, is believed to explain the com- 
mon failure to secure satisfactory results from control measures 
taken after the seedlings have come up and the disease has become 
noticeable. It is somewhat interesting to note that the data in the 



T)A-MPIXC-OFF IX FOREST NURSERIES. 



15 



table tend to confirm field observations that, a> compared with other 
species. Pinus resinosa is more susceptible to the Later forms of 
damping-off than to germination loss. 

Further indication that the killing of germinating seed before 
emergence may be important enough to help explain eases of poor 
germination is obtained by an entirely different method, as follows: 
At the Wind River Experiment Station of the United States Forest 
Service counts of the seedlings emerging and of those which later 
died were made on a number of untreated plats by forest officers, 
who kindly permitted the writer to use the data obtained. The 
counts were made separately on 10 plats each of noble fir (Abies 
nobilis) and silver fir (Abies eoncolor). The plats of each species 
had been sown with equal quantities of seed. It appeared on in- 
spection of the figures that the plats which showed the poorest emer- 




Fig. 8. — The area shown in figure 7 after the bed had been weeded and damping-off had 
practically ceased. (Photographed by S. C. Bruner.) 

gence were also the ones which suffered the most subsequent loss. The 
coefficient of correlation between the number of seedlings emerging 
and the percentage of subsequent loss in the same plats was found 
to be — 0.49±0.16 for the noble fir, and — 0.50±0.16 for the silver 
fir, an average of — 0.49±0.11 for the two species, confirming the 
conclusion drawn from inspection of the figures. In other words, 
poor emergence and heavy subsequent loss were in general associated. 
The simplest explanation of this association appears to be to suppose 
that both poor emergence and subsequent loss were largely clue to 
the same cause, namely, the damping-off parasites. Another possible 
explanation of the correlation would be to neglect parasites as im- 
portant causes of the poor emergence in certain plats and to suppose 
that the higher subsequent loss in such plats was due to heat injury, 
the less dense stands affording less shade to the bases of the seedlings 
composing it. As damping-off is in general so much more important 
than heat injury as a cause of death after emergence and the dif- 
ference in the degrees of shade between the plats with the denser 



16 BULLETIN 934, V. S. DEPARTMENT OF AGRICULTURE. 

and the plats with the thinner stands must have been very slight, this 
latter explanation has not much to support it. The data are believed 
to constitute further evidence of the importance of parasites in de- 
creasing the percentage of emergence in coniferous seed beds. That 
the effect of parasites on emergence should have been large enough 
in this case to make itself apparent on the face of the figures, despite 
the variations due to other sources, is especially interesting in view 
of the fact that the losses after emergence in these plats were not 
high. 

ECONOMIC IMPORTANCE OF DAMPING-OFF. 

The importance of damping-off in coniferous nurseries in Europe 
is indicated b}^ frequent reference to the disease in the literature. 
Biittner (25, 26) states that whole beds are frequently destroyed by 
it. Baudisch (9) speaks of the death of entire stands in many 
nurseries as the result of damping-off. In the United States Spauld- 
ing (137) considers damping-off a serious obstacle in forestation 
operations. Clinton (28, p. 348-349) reports serious damage to 
conifers in New England nurseries. The writer has found the dis- 
ease especially prevalent in nurseries in Nebraska and Kansas, a some- 
what unexpected situation in view of the relatively dry conditions 
prevailing there. A correspondent has reported heavy loss in seed 
beds in Texas. 

The economic importance of the disease in conifers is due in part 
to the rather heavy average losses experienced at many nurseries 
and in part to the irregular character of the losses. In one season 
losses may be negligible, while the next season the beds of certain 
species may be practically wiped out. Even without this element of 
uncertainty the losses experienced are expensive, because of the high 
cost of coniferous seed. The seed of some species costs from $3 to 
$5 a pound and seldom shows a germination of more than 60 per 
cent under nursery conditions. A loss of half of this 60 per cent from 
parasites, both before and after the seedlings break through the soil, 
is therefore a matter which deserves attention. The figures in 
column 8 of Table I, obtained by adding together those in columns 
6 and 7, show that the loss is frequently higher than this. At 
the nurseries at which control experiments have been conducted, the 
percentage of the seedlings in untreated beds which have been found 
by actual count damped-off after emergence is frequently more than 
50 per cent, in addition to the considerable but less accurately de- 
terminable loss indicated by the foregoing data as being caused by 
the parasites before the seedlings appear. 

It has been suggested by foresters and others that the net economic 
damage from damping-off is not as great as is indicated by the loss 
of seed and seedlings which it may cause. The argument is ad- 



DAMPING-OFF IX FOREST NURSERIES. 



17 



JOO 


/ 







2 


eo 




/ 


<?<? 


^-» 


60 


- / 

/ 

- 1 




30 
20 




20 


J 
1 

1 ^^ 


^* ^SULPHURIC ACID 

1 1 1 


IO 
SO 

<?o 


/ >S\SULPHURIC ACID 
<£~~T 1 1 1 


so 
^0 


~<3 


1 




30 




> 


30 


i 


zo 




1 - — 


so 


- / 


a /0 




^/ CX?/=>PSR SULPHATE 

1 1 1 


10 

zoo 
/eo 
/so 


/ ^/COPPER SALTS 


L /OO 

k eo 
\ eo 


3 


'"' 

1 
1 
t 

1 


*' 1 
t I 


V 00 

% 
S so 

»5 


> 


1 

1 \/ FORMAL DC HYDE 


30 
a-o 


J y/ SULPHURIC ACID 


\ 

1 

Ui 30 
kj 
^ so 


_Jf 


1 1 1 


eo 
so 
<?o 
30 
so 


' >r 1 1 1 


7 


/ 
1 
/ 
/ 

y / 


a 

— 1 
1 
1 


/o 


- 


/ s 

//SULPHURIC AC/D 

S*^| 1 1 


10 

ISO 


7 yT^ SULPHURIC ACID 
If 1 1 1 1 


ISO 


9 










100 


- 


j? 


/oo 


/' 


eo 


- 




eo 


t 

1 


eo 


- 


4 / 


60 


1 
1 


90 




» /I 

1 J 


40 


1 
/ 

/ S^SULPHURIC ACID 


zo 


1 
— f 

J 

r It 


I SULPHURIC ACID 
' 1 1 1 1 


zo 



10 



so &o so /o 20 30 

0<AY>S SINCE GERMIN/9T/ON 



<5k? SO 



Fig. 9. — Diagram showing the progress of damping-off in treated plats 
(solid line) as compared with untreated plats (broken line). Graphs 
1 to 4 represent Pinun ponderosa; graphs •"> to 8, /'. resmosaj graph 9, 
P. banksiana; and graph 10, plats each of which was half /'. nigra 
poiretiana and half P. sylvestris. Nurseries in Kansas, Nebraska. Minne- 
sota, and Michigan are represented. The relatively high total number 
damped off in the treated plats shown in two of the graphs is due to the 
fact that a large proportion of the seedlings in the untreated plats had 
been killed before they appeared above the soil surface. In both the 
eases in which the absolute number of seedlings killed was as great 
in the treated plats as in the controls, the percentage of the seedlings 
killed was nevertheless lower and the survival more than twice as good 
as in the controls. 

19651°— Bull. 934—21 2 



18 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

vanced that damping-off may be a valuable selective agent in nursery- 
grown stock for forest planting, eliminating the weaker individuals 
and thus insuring the vigor of the trees which go into the forest 
plantation. This is a possibility which must be considered. It is by 
no means certain, however, that escape from damping-off is correlate I 
with permanently superior vigor. It is believed that temperature, 
moisture, and other environmental factors, which as yet are very im- 
perfectly analyzed, together with the age of the seedlings and the 
presence or absence of virulent strains of the parasites, are much 
more important factors than inherent differences in individual re- 
sistance in determining whether or not seedlings are destroyed. Evi- 
dence from inoculation experiments and from field observation, sup- 
ported by data of the sort presented in Table I. indicate that damp- 
ing-off ordinarily does a considerable part of its damage by killing 
the sprouting seed before emergence from the soil, while the graphs 
in figure 9 show that of the loss which occurs after emergence in tin- 
treated beds a large part occurs very early in the life of the seedlings. 
Observation of the clean sweeps which the disease commonly makes 
in the immediate neighborhood of infection foci (figs. -\. 4. and 5) 
indicates that either before or just after the seedlings break through 
the soil none of them have any considerable resistance to the really 
virulent strains of the parasites, which are believed to be the ones 
responsible for the major share of the damping-off. 

Even if there should be found to be an appreciable selective value 
in damping-off, this would not be a valid argument against control 
by seed-bed disinfection for the following reason : The graphs show 
ing the course of damping-off in treated plats in figure 0. together 
with the decided differences in germination between treated and un- 
treated plats, indicate that the very early damping-off is more com- 
pletely controlled by disinfectants than the later damping-off. This 
early damping-off which the treatments so largely prevent is the 
part of the loss which has the least possibility of selective value. 
The later damping-off is rarely controlled at all thoroughly by disin- 
fectants. As shown by the graphs, it is often even heavier on the 
treated than on the untreated soil. It is the part of the loss which 
is most likely to have selective effect. At this stage beds are not 
taken clean, as earlier: only seedlings which are below normal re- 
sistance succumb. The damping-off in disinfected beds seems there- 
fore at least as likely to have true selective value as that which 
occurs in untreated beds. 

The only way in which the effect of damping-off as a selective 
agent can be positively determined will be to compare through sev- 
eral subsequent years the growth rate, or survival after transplant 
ing, of trees from beds which suffer seriously from damping-off 
with the growth of trees from the same lot of seed in seed beds in 



DAMPING-OFF IX FOREST NURSERIES. 19 

which the disease is either accidentally absent or is artifically con- 
trolled. Such an experiment is within the silvical father than the 
pathological investigative field. If it be found that there is some 
selective value in the action of the disease and that it is greater in 
untreated than in treated beds, it would still seem that a much more 
desirable and dependable selection could be obtained by discarding 
weak plants at the time of transplanting than by letting damping- 
off run uncontrolled in the seed beds. Damping-off is sometimes 
negligible and sometimes destroys practically all the seedlings in a 
given area, in neither of which cases can it have any material selective 
value. 

RELATIVE SUSCEPTIBILITY OF DIFFERENT CONIFERS. 

Biittner (25) writing of European conditions, states that exotic 
conifers are especially subject to damping-off. He includes fir, 
spruce, pines, larches, and cypress in this statement. He mentions 
the same subject in a later paper (20). Neger and Biittner (91) give 
a long list of different species of conifers from various parts of the 
Avorld with statements as to their susceptibility to damping-off. 
Beissner (11. p. 6T>G-0r>7), Neger (93), Clinton (28), Bates and 
Pierce (T), Boerker (13), and Tillotson (139, p. 69) have all given 
information on the susceptibility of different conifers. The data re- 
ported by Tillotson are drawn from reports by various officers of the 
United States Forest Service which he has compiled. While it is 
probable that the nurserymen who are responsible for most of his 
records have not observed the disease as closely as Neger and Biittner, 
the fact that their observations are mostly based on repeated seasons' 
work with large-scale seed beds of the species they report on makes 
their observations in some respects more reliable than the other pub- 
lished data. Neger and Biittner presumably worked in most cases 
with small beds of the various conifers on which they report, and the 
variations which they attribute to differences in specific resistance 
might easily in such case be largely due to accidental variation. The 
error which nurserymen are most likely to make in their notes on 
susceptibility is to underestimate the loss, especially for the small- 
seeded species. The seedlings of small-seeded conifers decay and 
shrivel so quickly after they fall that in taking notes at any one time 
only a small proportion of the total loss is visible. Frequent counts 
of dead seedlings are the only way by which the loss after germina- 
tion in such species can be properly appreciated. 

The data given by the authors mentioned in the foregoing para- 
graph, together with unpublished data obtained by personal observa- 
tion or from commercial and other nurserymen in the United States, 
are summarized in Table II, the source of each report being shown 
by letters signifying the authority. The unpublished data on two 



20 



BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 



nurseries yi Illinois and Minnesota were obtained from the nursery- 
men by Mr. R. G. Pierce and are indicated by the initial "P." In- 
formation obtained directly from the nurserymen by the writer is in- 
dicated by " N." For nurseries where the statements are based on 
the writer's personal observation rather than on the authority of the 
nurserymen, his own initial ("H") is given. Most of the writer's 
own estimates of relative susceptibility are based on a comparison 
of detailed counts of the damped-off seedlings in a large number of 
untreated plats at different times, as well as on observation. The 
nurseries on which Tillotson reported were all west of the Missouri 
River, most of them being in the mountain region. The reports in- 
dicated by "H" and "N" were mostly from nurseries east of the 
Rocky Mountains. In cases in which the data permit it, the species 
are classified as most susceptible, intermediate, least susceptible, or 
immune. In a number of cases, however, it is only possible to classify 
them as "more" or "less" susceptible. 

Table II. — Relative susceptibility to damping-off of different conifer species. 

[Figures in parentheses in this table indicate the number of nurseries from which the susceptibility noted 
has been reported by the observer to whom the preceding letter refers.] 







Reports of relative susceptibility.* 




Host species." 


Not 
sus- 
cep- 
tible. 


Least 
sus- 
cep- 
tible. 


Less 

than 

average. 


Inter- 
medi- 
ate. 


More sus- ,, , 
ceotible M "\' 
than the ' s f,^" 
average. u R ' 


Pinaceae (Abietoidese): 




('•) 
Nb.... 






























Nb... 














Bu, Nb. 








T 






Nb. 










Nb.... 












XI... . 












Nb 








Mi... 






















Nb. 








T 
















Nb.... 


N (2) 












Nb. 


Larix occidentals 







T 




T 










Ne 


Nb.. 










Nb.. 


P 


N.. 








T (2) 


H, N, Ne, Nb. 




P 


Nb .. 


H, Bu.... 




T. 

Ne, N 




Ne 

Ne 

N(2) 


Nb... 




N (2). 








Nb.. 




Picea pungens 






H, P.. 


Ne 


Picca sitchensis 






■ Nb. 

Ne Nb. 






Nb.. 




















N .. . 






Nb.... 
Nb.... 


N 

T 


P 


B, P, H B.. H. 




T 

T 


(2), T. 











n Host names for American species follow the usage in the publications and a later verba] communication 
of Mr. George B. Sudworth, of the CJhil <1 States Forest Service. For exotic species the standard Cyclo- 
pedia of IIoi'iinil!iin,\c\v York, 1916, edited by 1-. E. Bailey, is taken as the standard. The classification 
follows Saxton (UN). 

6 Symbols signifying the authority for the report: B=Boerker (13). Bu=Buttner (25, 26), Bp - Kales 
and Pierce (7), C=Clinton (28), H= Writer's estimate, N= Nurserymen s estimate (obtained by the writer I, 
Nb=Negerand Biittner (94), Ne=Neger (93), P= Nurserymen's estimate (obtained by Pierce), T=Forest 
officers' estimate (Compiled by Tillotson, 139). 

c Susceptibility to Phytophthorafagi. 



DAMPING-OFF IX FOREST NURSERIES. 



21 



Table II. — Relative susceptibility to dampmg-off of different conifer 
species < 'ontinued. 





Reports of relath e suscepl ibilitj . 


Host species. 


Not 
sus^ 
cep- 

tiblo. 


i ea i 
sus- 
cep- 
tible. 


Less 

than 

average. 


Inter- 
medi- 
ate. 


More ii - 
ceptible 
than the 

average. 


Most 
suscep- 
tible. 


Pinacese ( A.bietoidese) — Continued. 




Nb.... 














N 
















T 




T(2) 

T 
























P 








T 




T 




T 








Nb.-.. 


N, T(3).. 




Bp, N,T.. 




Pinus nigra poiretiana (Corsican pine) 


Bp . 




H... 










B 










Nb.... 










Pinus ponderosa (type not specified ) 

Pinus ponderosa (Eastern Rocky Moiui- 


T(3).. 


p 


Bp,N(4), 
T(6). 

II (1) 


Nb.... 


T(4) 

B 










B 














N (3) 




Bp, 11, T.. 
N 


B, H. 








P........ 








p 




11, B.N.. 
T(2) 


N 

H, 1'.. 


C, N'T... 
Bp,T(3).. 










N. 






B 








Nb.... 










Pseudotsugataxifolia (type not specified). 






N(3)T(3) 


11 


N, T(5)... 
B 






Nb... 




Pseudotsuga taxifolia (Northwestern 




B 




Nb.... 














Nli, P. 


Bu 
















9 

6 


17 
10 


51 
31 


23 

11 


48 
29 


15 




9 






Sciadopitoidese: 












Nb. 


Cupressacete (Cupressoideae): 




Nb.... 
Nb. .. 


T 




Bu 






















Nb.... 










Nb.... 












N. . 














Nb.... 






P 








N 














T... . 














II, P.. 
T... 


Nb... 











Nb.... 
Nb.... 


T 




















Nb... 










Thuja orientalis 


P... 






Nb.... 












Nb.... 




















Number of reports 


9 
39 


7 
30 


2 
9 


4 
17 


1 

4 







Sequoidem: 










B 














T 




Taxaceoe: 


V 

























The fact most evident in Table II is the extreme variation between 
reports, not only on closely related species but even on the same 
species. While it is, of course, possible that the obvious lack of a 
definite basis and method of comparison in most of the reports is 
responsible for most of this variation, it seems to the writer more 
probable that different species do actually vary in their relative sus- 
ceptibility to damping-off in different localities. Tn the first place. 



22 p.ri.LKTix 934, r. s. department of agriculture. 

the conditions which might increase resistance of one host might 
very easily decrease its resistance for a host with different environ- 
mental requirements. To illustrate by an extreme example, the 
pinon (Pinus edulis) of the arid or semiarid regiou might remain 
resistant in soils in which Picea engelmanni of the high mountain- 
stream bottoms or Picea mariana of the northern swamps might be 
low in vigor and easily attacked. In the second place, it is to be 
expected that species with a certain order of relative susceptibility to 
the parasites which predominate at one nursery may exhibit a very 
different order of susceptibility to the different combination of para- 
sites which might be prevalent in another locality. 

The only individual species on which there are a sufficient number, 
of reports and a sufficient agreement between the reports are the two 
common western spruces, Picea pungens and P. engelmanni, which 
(at least as compared with Picea excelsa) seem rather susceptible, 
and Pinus ponderosa., which (as compared with most of the other 
species of the Abietoidese) is to be regarded as generally more re- 
sistant than the average. Within each of the larger genera of this 
group it seems evident that susceptibility is extremely varied and 
that no statement as to the relative susceptibility of the genera them- 
selves can therefore be made. The only group generalization that 
is perhaps permissible is derived from the consideration of the 
Cupressoidese. In this group, out of 23 reports, 16 are in the " not 
susceptible " or " least susceptible " columns and only one indicates 
more than intermediate susceptibility. Of 163 reports pertaining 
to the Abietoidese, only 26 place them in the " not susceptible " or 
" least susceptible " columns and 63 in the classes of more than inter- 
mediate susceptibility. The general feeling among nurserymen 
seems to be that serious damping-off need not be feared among the 
cedars and their relatives. The data at hand tend to justify this 
confidence. 

CONTROL OF DAMPING-OFF. 

Early efforts to prevent damping-off were chiefly directed to the 
avoidance of excessive moisture in either the air or soil. A means 
to this end, which has been observed more or less by nurserymen for 
many years, both in the United States and elsewhere, is the applica- 
tion of small quantities of dry sand to the seed beds after the disease 
becomes noticeable (18, p. 166; 83). This is sometimes applied hot 
(101, p. 43^44; 145), though even this procedure does not result in 
very great advantage. Surfacing with hot sand can not always be 
counted on to give any measurable advantage over untreated beds (67. 
p. 3). The use of sand (25) or sterile subsoil (101) instead of ordi- 
nary soil in covering seed at the time of sowing has been advised. 
Johnson (82) did not secure satisfactory results with sand in tobacco 



DAMPlXd-OIT IX FOREST NtJRSEfclES. 23 

beds. Making the upper part of the bed to a depth of sex era] inches of 
recently dug subsoil appeared effective in a single test by Spaulding 
(137) and at four nurseries by cooperators of the writer in Inter 
tests, the results of which will be published elsewhere. The procedure 
is unfortunately rather expensive in large-scale work and under some 
conditions at least undesirable because of the poor subsequent growth 
on such soil. Excessive vegetable matter (45), imperfectly rotted 
manure (07), or green manures recently plowed under (43) have all 
been advised against as likely to favor the disease. The experience 
reported with conifers (07, 139) indicates that damping-off can be 
to a certain extent decreased by broadcast sowing as compared with 
sowing in drills. The usual recommendation of thin sowing to avoid 
the seed-bed disease of other plants lias also been made for conifers 
(67). Transplanting healthy seedlings from infected beds into new 
soil is recommended as a means of saving them from attack (11, 145). 
The writer's tests of transplanting at a Nebraska nursery gave no 
promise of economic value as a control method, although he is in- 
formed that it was successfully employed in a nursery in New Mex- 
ico. The time of sowing appears to have a relation to the amount of 
disease at some nurseries, but conditions in this regard evidently 
differ in different localities, so that' the best time to sow from the 
standpoint of avoiding damping-off must be determined separately 
by repeated tests at each nursery. For example, observations both 
by the nurserymen and the writer during several seasons at the 
Bessey Nursery, in Nebraska, indicate that fall sowing is an ex- 
cellent means of decreasing loss from damping-off in at least one 
pine species, and Retan (110) reports the same thing for a nursery in 
Pennsylvania, while at two Kansas nurseries and at nurseries men- 
tioned by Tillotson (139) fall-sown beds suffer more than those 
sown in the spring. 

Treatment of the seed with mercuric chlorid (25) or with copper 
sulphate (122) has been recommended. While it has been demon- 
strated (38) that a proper heat treatment of the seed will greatly 
decrease the damping-off in sugar-beet seedlings, this is explained 
by the fact that one of the most important parasites of the sugar 
beet is systemic and often present in the seed. There is no reason 
to believe that seed-carried infection is of any importance in conif- 
erous seed beds. The only advantage that could reasonably be 
expected from a seed treatment of conifers would be that which 
would come from the prevention of seed decay in the soil before 
germination starts, and this protection could be expected to be ef- 
fective only if a relatively insoluble disinfectant, such as Bordeaux 
mixture, was used. 

Soil treatment is the most direct and probably the most profitable 
method of attack on the disease. It is especially easy, lor tobacco 



24 BULLETIN 034, V. s. DEPARTMENT OF AGRICULTURE. 

seedlings (82) as well as for pines, to prevent by soil disinfection 
losses U'l'ore the seedlings appear above the ground. Heat disinfec- 
tion of seed beds lias boen frequently mentioned. Burning wood 
or litter on the surface of the beds before sowing, said by Gilbert (47, 
p. 36) to be a common procedure in preparing tobacco seed beds both 
in Italy and in parts of this country, has been recommended for 
coniferous seed beds by Biittner (25). The disadvantageous results 
sometimes noticed following the application of wood ashes to pine 
seed beds may prove an objection to this type of treatment in some 
of the nurseries. At a Nebraska nursery (67) moist heat proved only 
partly satisfactory, unavoidable reinfection having serious results. 
Steam disinfection, using the inverted-pan method commonly advo- 
cated for tobacco seedlings (10, 47, 81), has been reported by Scott 
(123) as successful at a nursery in Kansas. Gilford (46) found 
steaming with the inverted pan only partly satisfactory. It is not 
believed that it is likely to pay to install the necessary apparatus for 
steam disinfection at nurseries in nonagricultural districts where 
steam tractors are not available for temporary use. The hot-water 
soil treatment as used by Byars and Gilbert (27) is probably worth 
a trial at any nursery where damping-off is serious and fuel cheap. 
It may be that in some localities where steam or hot-water treatment 
of the soil is not sufficients effective, its efficiency can be increased 
by reinoculating the soil immediately after treatment with sapro- 
phytic molds and bacteria to provide maximum competition for 
parasites which come in from the outside. Tests of this procedure 
will be described later in the present bulletin. The value of char- 
coal has been emphasized by Eetan (109, 110). 

Chemical disinfection of the soil has also been employed. Sulphur 
has long been in use as a soil treatment against the damping-off of 
various plants (45, 111) in addition to its use in combating potato 
scab and onion smut. It was tested on conifers by Spaulding (136, 
137) in the form of light surface applications to the beds after ger- 
mination, but without decisive result. In later cooperative tests pow- 
dered sulphur raked into the soil before the sowing of the seed failed 
to indicate any large measure of value. Very finely divided forms 
of sulphur in various amounts and times of application are prob- 
ably worth some further test. 

Moller (90) and Sherbakoff (128) have reported the successful use 
of copper sulphate in combating attacks of Corticium on dicotyle- 
dons. In Johnson's experiments on tobacco seedlings (82, table 
3) copper salts and Bordeaux mixture were the only chemicals for 
which any value was indicated. Sherbakoff apparently used copper 
sulphate and other strong disinfectants chiefly to stop the extension 
of vigorously spreading damping-off foci by local treatment rather 
than as a general treatment for use over the beds. Such treatment 



I>A.M1MX(;-(>FF IN FOREST NURSERIES. 25 

would presumably kill all seedlings on the area treated, but would, 
of course. In- of considerable value in stopping at the outset sued 
mycelia as those which caused the damped-off area in figure 7. The 
procedure would be of practical value only in eases in which damp- 
ing-off was chiefly limited to a few large patches of this sort, a rather 
rare condition in conifers. 

Copper sulphate solutions have been used on pine seed beds at the 
time of sowing with considerable success at some nurseries (65, 67). 
Except in a nursery in which the soil contained carbonates, it has 
proved rather difficult to prevent injury to the pines. The trial of 
some such combination of copper sulphate and lime as was used 
by Spaulding (136) on the surface of pine beds before sowing, which 
apparently pi-evented the damping-off of lettuce in some unpub- 
lished pot experiments of Mr. J. F. Breazeale, is considered desir- 
able. Treating seed beds with ordinary Bordeaux mixture has also 
been recommended. Home (78) secured especially good results 
against Corticium vagum in tobacco seed beds by heavy applications 
of Bordeaux mixture, and Schramm (122) and Clinton (28) have 
obtained indications of its value as a spray in preventing the damp- 
ing-off of conifers. It is probably worth further tests in various 
amounts of application. In tests conducted by the writer in 1912 
and still unpublished, some advantage was indicated for Bordeaux 
mixture as a surface treatment after soil disinfection with acid. 
Zinc chlorid as a soil disinfectant has also been found valuable in a 
number of cases (65, 67), but it is more expensive and apparently less 
dependable than copper sulphate. 

Formaldehyde and sulphuric acid have been tested more fre- 
quently than other disinfectants. The use of sulphuric acid on 
coniferous seed beds was originated by Spaulding (136). The first 
intensive experiments with this acid were reported by the writer (63) . 
The first experiments with formaldehyde on conifers seem to have 
been in the early greenhouse tests of Spaulding (137), repeated in 
forest nurseries in 1907 by Jones (83) and Spaulding (136). Most 
of the experiments with these two substances have already been sum- 
marized (67). A report not mentioned in this summary is that of 
Schaaf (119, p. 88), who obtained favorable results with the acid. 
The great trouble with formaldehyde is its tendency to kill dormant 
seed. The length of time which must be allowed to elapse between 
treatment and sowing in order to avoid this killing varies with con- 
ditions. Formaldehyde is more expensive than acid and seems on 
the whole to have been less effective in disease control. Acid, on the 
other hand (applied just after the seed is sown, which is found to be 
the best time), on a few soils has caused injury to radicles, which it was 
at first thought could be prevented only by very frequent watering 
during the germination period; while in a few cases, when cold 



£6 BULLETIN &34, l'. S. DEPARTS i:ST OF AGRICULTURE. 

weather resulted in a long germination period, it has killed or in- 
hibited the germination of some of the dormant seed. All injury 
can be prevented by treatment a few days before sowing, followed 
by the addition of lime just before sowing, but lime used in this 
way has apparently destroyed :i considerable part of the value of the 
acid treatment against the disease. Further consideration of the 
data on which earlier papers (6-'5, 67) were based indicates that the 
apparent need for frequent watering during the germination period, 
which was required at a few of the nurseries where the first tests of 
acid treatment were made, ;is well as practically all of the germina- 
tion reduction, was due to the use of unnecessarily large applications 
of acid and that the trouble can be eliminated by determining by 
test the minimum quantity of acid which will be reasonably effective 
in each locality. If this can be done it should establish the acid 
treatment as the most profitable for general use of any of the 
methods of damping-off control which have so far been extensively 
tested. 

In view of the various parasites which may cause damping-off at 
different times and places and which vary greatly both in their 
means of dissemination and in their physiological qualities, it is not 
believed that any single disinfectant will be found entirely satisfac- 
tory at all nurseries. It is also unfortunately true that no one 
strength of treatment can be recommended for all nurseries. The 
quantity of acid to be used at any specified nursery will have to be 
determined by test at that nursery. A single test, no matter how 
well conducted, is not sufficient to serve as a basis for conclusions. 
However, a number of small-scale tests, made at different times and 
in different parts of the seed-bed area, should determine the best 
treatment for any particular nursery with a reasonable degree of 
certainty and with very little work. If the plats are equal in size 
and receive equal quantities of seed, all the nurseryman needs to do 
to determine the value of the treatments is to count the number of 
living seedlings on the different plats at the end of the season. The 
decrease in the number of weeds as a result of the use of acid is 
itself sufficient at a number of nurseries to pay the entire cost of the 
treatment. Detailed methods of application have already been pub- 
lished (67). The differing proportions of acid between which the 
best treatment will ordinarily be found to lie are 2 and 7 c. c. (one- 
sixteenth and one-fourth of a fluid ounce) of the concentrated com- 
mercial acid per square foot of seed bed, applied just after the seed is 
sown and covered. It should be dissolved in 500 to 1,000 c. c. (1 to 2 
pints) of water per square foot of bed before applying. The drier 
the soil before treatment, the more water should be used in dissolving 
the acid. 

No treatment applied after germination begins can have the maxi- 
mum value in controlling the disease, because the damping-off para- 



DAM H \i, nl'l' IX FOREST NURSERIES. 27 

sites frequently, it' not usually, do part of their work before the seed- 
lings appeal' above the soil. Furthermore, any treatment at all effect- 
ive against the disease is almost certain to hurt the seedlings if applied 
after the seed starts to sprout. 

Both the acid and copper-sulphate treatments which have been 
found useful in pine seed beds are of very doubtful value for most 
hosts other than conifers, as the angiosperms on which observations 
have so far been made are too easily injured by the disinfectants. 
The weeds in the nurseries have been injured or entirely kept from 
appearing by treatments which caused no injury to the pines. 

CAUSAL FUNGI. 

CORTICIUM VAGUM. 

Occurrence and parasitism. — Tn a recent publication (68) Corti- 
cium vagum B. and C. (C. vagum solard Burt. Hypochnus solani 
Pril. et Del., the common damping-off Rhizoctonia) has been reported 
on a number of conifers. Inoculation, reisolation, and reinoculation 
on pine have established its parisitism on this host beyond a reason- 
able doubt, though in these inoculations, as in most, if not all, the 
work which has been done with the fungus on angiospermous hosts, 
the cultures employed have been from plantings of diseased tissue 
instead of from single spores. The inoculation experiments have 
confirmed the field observations indicating that this fungus is fully 
as able to cause loss by destroying germinating seed below the soil 
surface as to cause damping-off of the better known type after the 
seedlings appear above the soil surface. 

An extensive list of angiosperms on which the fungus has been 
reported is given by Peltier (98). Cross-inoculations between the 
pines (G8), on the one hand, and potato (40) and sugar beet (38) 
have shown the same strains to be parasitic on both conifers and 
angiosperms and established the physiological as well as the morpho- 
logical identity of the fungus attacking pines with the common 
Corticium vagum. Now that Duggar (34) has offered strong, 
though not yet entirely conclusive, evidence of the identity of C. 
vagum with the European " vermehrungspilz " (the MonUiopsis 
aderholdil of Ruhland; 115) it is to be presumed that it is 
a cause of damping-off of conifers in Europe as well as in Amer- 
ica, though no reports of it on conifers have been so far en- 
countered in European literature. The Rhizoctonia reported by 
Somerville (132) on Pinus sylvestris and the Rhizoctonia strobi de- 
scribed by Scholz (121) as killing young Pinus strobus w r ere both 
on trees more than 4 years old, so that they had no relation to damp- 
ing-off. Furthermore, the first of these was apparently the old 
Rhizoctonia violacea, now known as /?. crocorum (i?. medacaginis) , 
a fungus entirely distinct from Corticium vagum, probably belong- 



28 



BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 



ing to an altogether differenl group of fungi and not known to c;\u^' 
damping-off of any host. Rhizoctonia strobi is not sufficiently de- 
scribed to allow determination of its identity. 

Variations in virulence. — In the inoculations earlier reported on 
conifers, different strains of Corticium vagwm were said to vary 
greatly in virulence (68). Further examination of the data on 
which this statement Mas based yields confirmatory evidence. Part 
of this evidence is shown graphically in figures 1, 2, and 10. The 
experiments on which these graphs were based involved at the time of 
seed sowing the addition to the soil of apparently pure cultures of 
C. vagum. Throughout each experiment the different units received 



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Fig. 10. — Diagram showing the relative activity of different strains of Corticium vagum, 
as indicated by the number of seedlings appearing in inoculated pots. Explanation of 
symbols : O =Strain 147, from spruce seedlings, Washington, D. C, 1910; V =strain 
213, from sugar beet seedlings, Washington, D. C, 1911 ; H=strain 230, from Elaeagnus 
sp., Kansas, 1913 ; □ =strain 233, from the same lesion as strain 230. Strains re- 
isolated from these, the results of which appear in experiments Nos. 71 and 72, are 
indicated by the same signs as the original strains used in the inoculations from which 
they were taken. The original strains in experiments Nos. 71 and 72 are indicated 
by arrows. 

equal quantities of seed, and the culture substratum used in inoculat- 
ing was the same for all strains. Experiments 36, 45, 47, 49, and 51 
were conducted on plats in out-of-door drill-sown beds, experiment 
36 on an alkaline soil, all of which had been heated in a moist con- 
dition at a temperature of not less than 80° C. for not less than 10 
minutes, 2 and experiments 45, 47, 49, and 51 on a sand which had 



2 This temperature is probably high enough to eliminate dainping-off organisms. Tests 
by Dr. Theodore C Merrill indicate that the three most virulent parasites so far 
worked witli are killed by placing agar tube cultures for 10-minute periods in water ai 
the following temperatures : Pythium debaryanum, G~>° C. ; Corticium vagum, 50° C. for 
mycelium and (>o° c. for sclerolia; Fusarium moniUforme, 70° C. Both the Pythium 
and Fusarium cultures contained spores. The possibility of the survival of oospores 
which would not be capable of germination for several months was apparently eliminated 
by the writer, who retained Dr. Merrill's heated Pythium tubes and made final transfers 
from them 7i months after heating, still without securing growth. Plenty of typical 
oospores were present in the part of the heated culture from which transfers were 
made. 



DAMPIXC-OFF IX FOREST NURSERIES. 29 

been treated with sulphuric acid followed later by lime. The other 
experiments included in the graphs were on autoclaved sandy loams 
in pots in the greenhouse. Tn these graphs are included all of the 
results in which the same groups of strains were used repeatedly in 

different experiments. In figure 1. the values plotted for experi- 
ments 36, 49, and 51 are for the number of seedlings which appeared 
above around, the heavy inoculations and favorable conditions for 
damping-off in these experiments being such that even weak strains 
caused heavy losses and the survivals therefore do not give differ- 
ential results. Comparison of the survival data in the other experi- 
ments in figure 1 with the emergence data for the same strains in 
that figure and in figure 10 indicates that the strains best able to 
reduce survival are also the ones best able to reduce emergence. 

While the data presented in the graphs are not entirely consistent, 
it is very evident from them that strains 147, 213, and in a lesser 
degree 206 were regularly more virulent than most of the strains in 
tests conducted several years apart on different species of Pinus. 
It is also evident that certain strains of 1S6 and ISO which appear 
in figure 2 are quite regularly of low or doubtful virulence. Strains 50, 
183. 19-2. -ill. 212, 230, and 233. whose virulence is apparently inter- 
mediate, show a greater variability. In experiments 36. -45. 47, 49, 
and 51, in which conditions especially favor parasitism, they may 
cause practically as serious loss as the regularly virulent strains, the 
best differential results being shown in experiments in which the 
disease is less favored. The apparent variation in the relative viru- 
lence of such strains in different experiments may, of course, mean that 
their virulence is differently affected by different conditions. It 
seems rather more probable that the variation in relative activity is 
to be classed as accidental variation, necessarily great with small 
units which are subject to numerous uncontrollable variables. It 
seems entirely possible, however, that part of the observed differences 
in relative activity may lie due to differences, not in virulence, but in 
the ability of the different strains to maintain themselves saprophyt- 
ically in different soils during the period between inoculation and 
the commencement of germination. For example, strains 230 and 
233 came from a nursery in southwestern Kansas in which the soil- 
acidity exponent, as determined by Dr. L. J. Gillespie, of the United 
States Bureau of Plant Industry, is 8.4. It seems entirely possible 
that these strains, rather strongly parasitic in some of the experi- 
ments, including an experiment on the soil from which they w r ere 
taken, might prove less able than strains from some other habitats 
to maintain themselves on some of the eastern soils used in the green- 
house tests. The source of strains 230 and 233 was furthermore a 
locality where high soil temperatures are to be expected. The fact 



30 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

that experiments 71 and 72. in which they showed the least virulence, 

were conducted in a colder greenhouse than any of the other tests 
may have had something to do with the lower activity indicated for 
these strains. Variation in the temperature requirements of different 
strains in accordance with the temperature of the source locality has 
already been demonstrated by Edgerton (35) for one of the anthrac- 
noses. It is hoped later to determine the temperature and acidity 
preferences of these two strains as compared with the others used. 
It should be noted that the consistently weak strain No. L89 (fig. 2). 
was abnormal in habit, lighter brown, and produced fewer sclerotia 
than typical strains. The other strains appearing in the graphs 
showed no conspicuous morphological or cultural differences that 
were identical. The only other strain which was noticeably abnormal 
in culture was one from pine seedlings in Kansas, intermediate in 
habit and color between No. ISO and the typical strains and indicat- 
ing little more virulence than Xo. 180 in the lV\v experiments in 
which it was used. It does not appear in figures 1 and 2. but was 
included in the experiments reported in the following paragraph. 
Peltier (00) believes low sclerotium-forming capacity to be a sign 
of degeneration and low virulence; the writer's experience agrees 
with his as to virulence, but these two strains showed no other evi- 
dence of lack of vigor. 

As a further check on the reality of the apparent differences in 
virulence between different strains, all of the original strains avail- 
able at the time, a total of 20, were used in the practically duplicate 
experiments 71 and 72 and the relative survivals of the same strains 
in the two series mathematically and graphically compared (fig. 11). 
The very decided correlation between the two experiments indicated 
by the graph has a coefficient 3 of 0.813±0.042, nineteen times its 

3 The correlation coefficient, a very useful thing for many kinds of biological work, 
which unfortunately has received little attention from plant pathologists, is explained 
by Secrist (124, p. 43 et seq.) and the process of computation described (124, p. 453—467). 
A shorter method of computation is given by E. Davenport (30, p. 465—467) : the 
example he gives is of a series with a large number of varieties, in which the correlation 
table is employed. Davenport's method is, however, just as useful in such a case as 
this, in which the number of varieties is too small to make the formal table advantageous. 
In such a case the computation should be arranged as by Secrist (124. p. 460—461), but 
the guessed rather than the lino mean used and Davenport's formula employed. If the 
coefficient is i- 1. the correlation is perfect: if it is there is no correlation,, and if 
— 1. perfect negative correlation. The significance of a coefficient less than 1 is judged 
from iis relation to its probable error. King (84), in an excellent discussion of cor 
relation, uives rules for judging the degree of significance of the coefficient. The 
correlation coefficient has its greatest potential usefulness in examining apparent causal 
relations. It is soused in connection with the relation between the hydrogen-ion exponent 
and damping-off in considering figure 1 2 of the present bulletin. Interexperimental, 
or, as Harris calls them, " interannnal " correlation coefficients of the sort used for 
these Corticium strains have been used by Norton (00, p. 51) in measuring the constancy 
of rust resistance of asparagus strains, by Harris 1 54 l in demonstrating the constancy 
of differences in various characters between strains or individuals, and they appear to 
be useful for this purpose jn the present case. 



DAMPING-OFF IX FOREST NURSERIES. 



31 



probable error. Peltier's results permit similar correlations for the 
18 strains common to his experiments 1 and 1A on carnation cuttings 
and for the 22 strains common to experiment 1 on cuttings and ex- 
periment 2 on seedlings. The coefficients found are decidedly lower 
than those obtained from the experiments on pine. 0.51 ±0.117 for 
the experiments on cuttings and 0.36±0.124 for the comparison 
of the results on cuttings with the results on seedlings, hut neverthe- 
less indicate some interexperimental correlation for the same strains 
and therefore inherent differences in parasitic ability between the dif- 
ferent strains. 




STRftf/VS OFCORT/C/UM l/rfGUM 

Pig. 11. — Diagram showing the comparative virulence of 20 strains of Corticium vagum 
in two successive inoculation experiments on Pinus resinosa. The results in experiment 
Xo. 71 are shown by the solid line, the strains being arranged from left to right in the 
order of descending virulence indicated by the number of seedlings surviving in this 
experiment. The results from the use of the same strains in experiment Xo. 72 are 
shown by the broken line. The obvious correlation between the two curves (coefficient 
0.81±0.04) indicates a real difference in virulence between the different strains. The 
strains indicated by the underscored numbers are original strains and those not under- 
scored reisolations from the original strains in earlier inoculation experiments on pine 
seedlings. 

In the work on pine seedlings, with the possible exception of 
strains 230 and 233, there was no evidence of attenuation in arti- 
ficial culture. Strains 147 and 213, isolated in 1910 and 1911, re- 
spectively, seemed as strongly parasitic in experiments 71 and 72 
(1917 and 1918) as any of the five strains isolated in 1916 or the six 
strains isolated in 1915. 

Of the 20 strains above mentioned, three pairs were isolated under 
such conditions and showed such later agreement in performance as to 
indicate their individual identity. For the purposes of considera- 
tion in the following paragraph, the One of these probably duplicate 
strains which happens to have the higher number was eliminated from 
each pair. The survival figures in pots inoculated with the 17 strains 



32 



BULLETIN 934, r. s. DEPARTMENT OF AGRICULTURE. 



remaining, giving the mean of the results in experiments 71 and 72, 
are shown graphically in figure 13, together with the results of some 
of Peltier's experiments in which other strains were used. Per- 
centages of seedlings damped-off after germination are not included 
in these and most of the other data on pines because the most viru- 
lent strains often entirely prevent germination, and no value for sub- 
sequent loss is obtainable. The grouping of most of the writer's 
strains at the least virulent end of the register (that is, the one with 
the highest number of living seedlings) is of some interest. The 
distributions based on the two experiments considered separately 



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Fig. 12. — Diagram showing the relation between damping-off of conifers (broken line) 
and soil acidity (solid line). The acidity of soil samples from the different. nurseries, 
determined by Dr. L. J. Gillespie, is reported as P H 7, indicating approximate neutrality 
while P a 6 indicates ten. and T H 5 one hundred times as great a hydrogen-ion concentra- 
tion as I'h" ; therefore the lower the hydrogen-ion exponent line, the greater the acidity. 
The seriousness of damping-off at each nursery is on an arbitrary scale in which nurseries 
with negligible loss are rated as 1, and the nursery which suffered most is rated as 10. 
These values are estimates, though for some of the nurseries extensive counts were 
available on which the estimates were based. 

agreed Aery well in this grouping. The minor group at the end of 
extreme virulence is not taken to indicate an actual grouping but, 
rather, an artificial one, due to the fact that both the strongest 
strains and some less strong were thrown into the same group by 
the lack of additional seedlings for the stronger strains to kill. This 
lack of additional seedlings constituted a limiting factor. In other 
words, conditions favored damping-off even in these two experiments 
too much to permit completely differential results for the more viru- 
lent strains. Despite this artificial limit preventing the full vari- 
ability becoming evident, the coefficient of variability of the survivals 



DAMPING-OFF IN FOREST NURSERIES. 33 

had the high value of 63±9.7 per cent. The graph indicates also a 
decided, though less extreme, degree of variability for Peltier's 
strains on carnations; the survivals for the 18 strains which he used 
in both of his experiments on cuttings have a variability coefficient 
of 29±3.5 per cent and the , _ ) -' , > strains in experiment 2 on seedlings 
55±G.9 per cent. 



/=>YTH/UM DEB/9RY/9/VI/M 



• oo 



CORT/C/UM VrtGUM 
Wtf/TEftS &TRP/NS ON R//VE 



D □ 

□ CD □ DD □ □ D [TDDD 



cP 



nP&T/ERS STR/9/NS O/V ' CfiftNtiT/ON CUTT/NGS 

an □ 

ODDD DD D DD D DD □ 



PELT/ER'S STR/9/NS ON CRRHRTIOM SEEDL/NGS 

a a 

DD D DDD DD 

DD DDDDDDD DDDD 



O 20 40 60 eo /oo 

PI/9MTS &ORI//V//V0 PER /OOJN CONTROLS 

Fig. 13. — Diagram showing the results of inoculations with 17 strains of Corticium vagum 
and 3,"> strains of Pythium debaryanum, arranged in decreasing order of virulence from 
left to right, as indicated by the survivals in pots of pine seedlings artificially inoculated 
with them. The Pythium results represent the mean survivals in 5 pots inoculated with 
each strain in each of experiments Nos. (!(!, <i7, and 6S. Each point located is therefore 
based on the results in 15 pots, 10 of I'intis banksiana and 5 of P. resinoaa. The 
Corticium results on pine represent 5 or G pots each, in two experiments (Nos. 71 and 72) 
on P. rcsinosa. The outline circles represent /'. debaryanum strains from East Tawas, 
Mich. ; the solid circles represent strains from other localities. The second row of 
squares shows the sum of the results in Peltier's experiments Nos. 1 and la (99, his 
table 3). The lowest row of squares shows his results in experiment No. 2 (his 
table 4). 

The strains represented in figure 13, as used on pine, include 1 
from bean, 2 from potato, 1 from sugar beet, 1 from Elaeagnus, 2 
from Picea engelmcmni, and 10 from Phi us resinosa, P. ponderosa, 
and P. banksiana. Two were from Washington, D. C, 2 from New 
York, 1 from Ohio, 4 from Michigan, 4 from Minnesota, 1 from Ne- 
braska, 2 from Kansas, and 1 from California. The sources of these 
strains are widely distributed both as to host and locality; they are 
19651°— Bull. 934—21 3 



34 BUUL.BTIN 934, U. s. DEPARTMENT OF AGRICUL.TTJEE. 

rather more representative of the country as a whole geographically 
than the strains in the larger of Peltier's experiments, though less 
representative as to host sources. The number is too small to justify 
conclusions as to the proportion of Corticium vagum strains which 
can be expected to prove strongly virulent on pine. The data are 
offered merely as a beginning, to which it is hoped experimenters 
with other strains of C. vagum will make additions. 

In addition to the strains used in these two experiments, several 
others which had been lost or for other reasons could not be included 
in both the final experiments had been previously tested on pines in 
earlier experiments. Of these, 6 strains, 1 each from alfalfa, sugar 
beet, Pseudotsuyc taxifolia, Pinus htniksiana, P. resinosa, and /'. 
strobus, gave indications of low virulence on the pines; 3 strains, 1 
each from sugar beet, Pin us sylvestris, and P. ponderosa, gave indi- 
cations of rather high virulence; while another strain from P. ponde- 
rosa indicated an intermediate ability to attack pine. Combining 
these strains with those represented in figure 13, there are data on 27 
original strains, of which 8 are roughly classed as strongly virulent 
on pine seedlings, 14 as weak, and 5 as intermediate. 

Edson and Shapovalov (40) have conducted inoculation experi- 
ments on potato stems with G of the Corticium strains which had 
been used on pine, including the 5 strains mentioned by them on page 
218 and their strains R. XV (the writer's strain 192 of fig. 2) on page 
215. Strains 147 and 724, which had proved the most destructive 
in the inoculations on pine, appeared also rather strongly virulent on 
potato. Strain No. 186, originally from potato, which had given no 
definite evidence of parasitism on pine, also proved unable to cause 
lesions on the potato stems. The remaining 3 strains, all of inter- 
mediate virulence on pine, gave results on potato which were less in- 
dicative of agreement with the order of virulence on pine. The data 
suggest that strains strongly parasitic on potato are likel} r to be 
strongly parasitic on pine, and vice versa, but the agreement between 
their results and the writer's is not sufficiently complete to establish 
the point. 

FUSARIUM SPP. 

Fusarium is often found on or in damped-off seedlings (24, 46, 60, 
94, 120, 137, 141, 142). The early inoculation experiments, conducted 
in the main with strains not sufficiently described to allow their iden- 
tification, have been recently summarized (68, p. 537), together with 
descriptions of inoculation experiments on pine seedlings with four 
commonly recognized species of Fusarium. These, though not fol- 
lowed by reisolation, gave rather definite evidence that Fusarium 
moniliforme Sheldon was decidedly parasitic and F. solan'i less 
strongly so. Fusarium. ventricosum Appel and Wollenw. was indi- 



DAMPING-OFF IN FOREST NURSERIES. 35 

r:itcd ns more strongly parasitic than F. solani, but in a single test 
only and with a culture of doubtful purity. Fusarium acuminatum 
E, and E. gave no evidence of parasitism. These results agreed with 
those of Spaulding (137) in indicating that the ability to attack 
seedling conifers is not limited to a single species of Fusarium and 
that /•'. monUiforme is one of the more virulent. The statement by 
Hartig (61, p. 147— ir>o) that a Fusariumlike fungus was able to cor- 
rode the young epidermis of pine seedlings has already been men- 
tioned. 

PYTHIUM DEBARYANUM. 

Pythium debaryanum llesse (Artotrogus debaryanwm Atkinson, 
Luczdium pytJiioides Lohde) has been known since 1874 (74, 86) as 
a common cause of damping-off of various angiosperms. The first 
known observation was made by De Bary about 1864 (74). Despite 
the large number of hosts on which it has been listed, its parasitism 
has been definitely established on few. Peters (100) has successfully 
inoculated sugar beets with pure cultures ; at least part of his strains, 
including presumably part or all of those he used in inoculation tests, 
were obtained from single spores. Edson (38) working with the 
same host, reisolated the fungus from inoculated seedlings, and made 
reinoculations with it. Both find it able to cause root sickness of 
plants not attacked early enough to be killed outright. Johnson 
(82) and Knechtel (85) have caused damping-off of tobacco seed- 
lings with it, and the former reported it also able to persist in the 
cortex and kill the lower leaves of tobacco plants which survived 
attack. The fungus has long been reputed parasitic on potato tubers 
and has now been found by Hawkins (70) to be the chief cause of 
the rot known as "leak" in California. Peters (99) made success- 
ful inoculations with pure cultures on cuttings of Pelargonium. 
Most of the reports of parasitism, however, have been based on 
microscopic examination or more or less crude inoculation experi- 
ments. Noteworthy among the latter are those reported by Hesse 
(74) on Camelina sativa in the original description of the fungus. 
These were made before pure-culture technique had come into use 
with fungi, but were so thoroughly checked by microscopic obser- 
vations at every step in the process that they must be admitted as 
very good evidence of the parasitism of the fungus. A number of 
reported angiospermous hosts are listed by Butler (23), Voglino 
(143), and Johnson (82, p. 34, footnote, and p. 35). Reinking (107) 
recently reported Canica papaya as attacked. A host which the 
writer has not found in the literature is rice, found by Dr. Haven 
Metcalf seriously attacked in the seedling stage in a field in South 
Carolina. A second apparently new host for the fungus is fenu- 
greek {Trigonella foenum-graecum) . The writer found oospores 
tvpical of Pythium debaryanum in the tissues of damped-off seed- 



36 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

lings received by Prof. William T. Home, from Sonoma County, 
Calif., with the statement that the disease was seriously affecting 
the stand. The fungus was easily isolated, and the results of suc- 
cessful inoculations on pine with the cultures obtained are included 
in Table V (p. 47). A fungus resembling P. debaryanum was 
also found in damped-off cowpea (Vigna sp.) seedlings grown in 
rotation with pines at a Nebraska nursery. 

Pythdwn equiseti Sadebeck, reported as parastic on the prothallia 
of Kijii'ix' turn arvenxt\ was successfully used by Sadebeck (117) in 
crude cross-inoculations direct from E. arvense to potato tubers. 
I>e Bary (5) reversed the direction of the experiment between 
eryptogamous and phanerogamous hosts by successfully inoculating 
prothallia of Equisetum arvense with Pythium debaryanum directly 
from diseased Lepidium seedlings. He also secured positive results 
on prothallia of the fern Todea africana by the same method. The 
Equisetum prothallia he found to be especially favorable media on 
which to develop Pythium debaryanum,. Fischer considers the 
fungus found by Bruchmann (17) and Goebel (49) on prothallia of 
Lycopodium sp. as probably identical with P. debaryanum. A care- 
ful reading of the original articles is sufficient to show that the sym- 
biotic fungus wdiich they described was an entirely different or- 
ganism. Saprolegnia schachtii and Sporodospora jungermanniae, re- 
ported on two of the Hepa'ticse, are of doubtful position (42, p. 403), 
though Butler ('23, p. 89), after a survey of the literature, apparently 
favors the view that the former is distinct from the damping-off 
fungus. De Bary (5) reported Vaucheria and Spirogyra apparently 
immune against P. debaryanum. 

Early references to Pythium debaryanum in connection with 
gynosperms seem to have been based on the probability that it 
would be found to be the cause of damping-off in conifers (6 ; 97 ; 134, 
p. 27). The first actual finding of the fungus in any gymnosperms 
of which the writer is aware is indicated by a label marked Pythium 
debaryanum in the handwriting of Mrs. Flora W. Patterson on a 
package of coniferous seedlings from a New York nursery collected 
in 1904. 4 The seedlings, judging from the several rather long cotyle- 
dons and the fact that both cotyledons and primary leaves are denticu- 
late, are probably of one of the species of Pinus having medium-sized 
seed. In 1908 Dr. R. J. Pool, of the University of Nebraska, and his 
student, Mr. H. S. Stevenson, obtained in culture from damped-off 
coniferous seedlings a nonseptate fungus which was probably 
Pythium debaryanum, but which formed no distinctive spores on the 
media on which it was grown. A year later the writer obtained the 
fungus from pine seedlings at the same nursery and reported it as 

4 In the Office of Pathological Collections, United States Bureau of Plant Industry. 



DAMPING-OPE IX FOREST KTURSERIES. 37 

parasitic on pines in preliminary inoculation experiments (62). In 
L910 Spaulding (b'57) found it on spruce in NeAv York, and Hof 
niann (76) later made successful inoculations on both pine and spruce 
seedlings, using P. debaryanum, cultures both from aerial trap plates 
and from recently damped-off seedlings of cabbage, radish, and 
Russian thistle {Salsola tragus). Hofmann's work, detailed notes of 
which the writer has been permitted to examine, was done with cul- 
tures which were contaminated by molds, but was checked up by 
microscopic examination of the lesions resulting, which showed the 
affected tissues filled with nonseptate hyphse. His results are taken as 
a rather strong indication that /\ debaryanwm, attacks spruce as well 
as pine and that the fungus attacking conifers is physiologically as 
well as morphologically identical with that causing the damping-off 
of angiosperms. 

There thus appears from the literature to be reason to believe that 
Py tit iu m debaryaw/um is parasitic on representatives of two groups 
of the Pteridophyta and on gymnosperms, as well as on various 
monocotyledons and dicotyledons, a range of hosts not only remark- 
able but perhaps unequaled in our present knowledge of plant 
parasites. Final published proof of parasitism seems to be available 
for three or four species of dicotyledons only. Additional inocula- 
tions on conifers with strains isolated from various other hosts are 
reported in the present bulletin. Some of the detailed evidence neces- 
sary for complete proof of the parasitism of the Pythium on conifers, 
lacking in experiments previously reported because of the doubtful 
purity of the cultures used and failure to reisolate and reinoculate 
with the organism, is also given here, together with evidence of the 
ability of the parasite to cause root sickness of pines too old to suffer 
from damping-off. 

Descriptive data of interest on Pythium debaryanwm have been 
supplied by Hesse (74),. De Bary (5), Ward (144), Miyake (89), 
Butler (23), and Butler and Kulkarni (24). An important contri- 
bution to the physiology of the fungus and the factors controlling its 
passage through the tissues of one of its hosts has recently been made 
in the previously mentioned paper of Hawkins and Harvey (71). 

IDENTITY AND ISOLATION. 

The fungus in the writer's cultures referred to Pythium debary- 
anum Hesse has been so called for the following reasons : 

(1) The morphological characters agree with those described and figured for 
Pythium debaryanum by other workers and with those of strains obtained from 
Dr. H. A. Edson under this name. 

(2) The absence of zoospores in the writer's cultures agrees with the experi- 
ence of others with PytMum debaryanum (2, 5, 23, 24, 38, 100), nil workers 
with pure cultures having obtained zoospores infrequently, if at all. The 
earliest work by Hesse (74) in which zoospores were apparently produced 



38 BULLETIN 934, l\ s. DEPARTMENT OF AGRICULTURE. 

readily :ii certain times of the year was done before the development of pure- 
culture methods. Water cultures kepi in the dark and in the Light, at constant 
and at varying temperatures, with nutrient substrata consisting of steamed 
or outoclaved fragments of potato, carrot, sweet potato, turnip, sugar beet, corn 
meal or rice, nutrient agar, sugar-beet seedlings, and insects have all produced 
only sexual fruiting bodies and chlamydospores (the so-called conidia). 

(3) The successful cross-inoculations, those which Edson (38) used on sugar- 
beel strains and the writer had found parasitic on pine and had used on pine, 
strains winch Hawkins had found parasitic on potato tubers and Edson on' 
sugar beets, confirm the work of ELofmann (77) in indicating that the Pythium 
which causes the damping-off pine is a parasite on entirely unrelated species 
of host plants, a commonly recognized characteristic of Pythi/wm debaryanum. 

The organism is easily isolated from recently damped-off conifer- 
ous seedlings or from soil direct by placing the seedlings or a lump 
of soil at the edge of a Petri dish of solidified prune agar and transfer- 
ring to tubes mycelium from the advancing edge of the resulting 
growth. It has been found in or obtained from damped-off conifers in 
California. Kansas,Nebraska,Minnesota,and the District of Columbia, 
as well as in cultures made by Mr. Glenn G. Hahn in Michigan. Plcea 
engd niduni, P. sitcTu /<*is, Tsuga mertensiana, Pinus nigra austriaca, 
and Pseudotsuga taxifolia tire the coniferous hosts from which cul- 
tures of Pythium debaryanum have been obtained. It has been iso- 
lated directly from soil not only in coniferous seed beds but from 
open grassland in California not adjacent to any seed bed or culti- 
vated crop. Unless Mucor is abundant, Pythium is commonly ob- 
tained in apparently pure condition on the first transfer from the 
plate, as prune agar appears unfavorable for most bacterial growth 
while allowing rapid spread of the Pythium. On media made from 
prunes which taste sweet and with a total gross weight of not more 
than 40 or 50 grams per liter of medium, the Pythium will make a 
rapid growth, often extending radially 1 mm. per hour at tempera- 
tures in the neighborhood of 22° C. and produce both chlamydospores 
and oospores. A less valuable medium for isolation work, but more 
convenient for subcultures than any other which has been tested, is 
autoclaved corn-meal agar. The growth is not luxuriant, but spores 
are always formed and the cultures seem to be as long lived as those 
on any other medium, retransfer being rarely necessary more often 
than twice a year. Much stronger growth and more abundant fruit- 
ing is obtained on such media as sugar-beet or rice-stem agar, but 
the leathery surface of the culture on such media makes transferring 
difficult. On rice grains, corn-meal mush, beef agar, and on corn- 
meal agar plus 2 per cent dextose or sucrose no spores are formed 
and the cultures are short lived, though growth is heavy and on 
the last-named medium extremely rapid. On agar containing the 
juice from sour prunes or on corn-meal agar prepared without sub- 
jecting it to the high temperature of the autoclave, both growth and 
fruiting have been very poor or even lacking. 



DAMPING OFF IX FOREST NURSERIES. 39 

In both artificial cultures and in the tissues of coniferous and 
dicotyledonous hosts the numerous strains observed showed no con- 
spicuous differences in the size or other characters of the spores pro- 
duced, though noticeable and constant abnormality was found in one 
strain in the readiness with which spores were produced and in two 
strains in the ratio between chlamydospores and oospores in agar 
cultures. In the first-mentioned strain, obtained from pine in 
Kansas, and in cultures reisolated from seedlings inoculated with it, 
both chlamydospores and oospores are produced tardily and so 
scantily that it is often difficult to find them. In most strains, on 
the other hand, almost the entire contents of the mycelium are 
promptly emptied into the spores as soon as the limits of rapid vege- 
tative growth are reached. In another abnormal strain from pine 
from the same locality, and in still another furnished by Hawkins 
from a California potato, chlamydospores are produced in large 
numbers, but oospores are few. In many other strains, including 
several from California, the opposite condition obtains, oospores in 
plate cultures being decidedly more numerous than chlamydospores. 
These peculiarities of particular strains seem to be fairly constant 
characters, the first abnormal strain mentioned having been under 
observation for more than three years without any change in its tend- 
ency to scanty fruiting, and the low ratio of oospores to chlamydo- 
spores having been constant during the shorter periods over which 
the observation of the other strains extended. In view of the small 
variation between different strains in the matter of speed of growth, 
a purely vegetative character, this variation in reproductive habit 
is somewhat surprising. The strain which produced spores infre- 
quently was unquestionably parasitic, though it killed fewer seed- 
lings than the average Pythlum deharycunum strains. The strains 
with the high ratio of chlamydospores were both of at least average 
virulence on pine. 

Oospores in the strains the writer has had in culture, whether ex- 
amined in agar, in water cultures, or in root tissues, have ordinarily 
been somewhat larger than the diameter of 14 or 15 to 18 pi given in 
a number of the descriptions. The maximum range has been 12.8 
to 20.6 [j., the same strain sometimes being well down within the usual 
size range and sometimes ranging from 17 to 20 \i. The largest 
oogones observed were 26 \i in diameter. Various stages of fertili- 
zation are shown in Plate I, figures 2 to 4. Chlamydospores attain 
a maximum diameter in the case of the limoniform intercalary forms 
of 32 [x, and spherical chlamydospores sometimes reach a diameter 
of 28 [jl. There is no lower limit for these bodies, as under unfavor- 
able conditions — e. g., in sour-prune agar — they are sometimes all less 
than 15 [/. in diameter, and the smaller ones are little larger than the 



40 BULLETIN 934, 0. S. DEPARTMENT OF AGRICULTURE. 

hyphse which bear them. Both oogones and chlamydospores may be 
either termina] or intercalary. 
The normal hyphse are large, varying from 3 to 7 \j. and sometimes 

more in diameter. Typical hyphse, showing the false septa developed 
at the boundary of the protoplasm and the portions of the hyphse 
which have been evacuated in the extension of the younger parts, are 
shown in Plate I, figure 1. At points at which the ends of hyphse come 
in contact with the glass of tin 1 culture dish, peculiar contact swell- 
ings are produced (PL I, figs. 5 to 7), much the shape and size of 
antheridia, but not walled off from the adjacent hyphse and having 
no apparent significance in the life history of the fungus. These are 
not always terminal (PL I, fig. 8). It is noteworthy that Hesse de- 
scribed contact swellings at the tips of the hyphse just before pene- 
trating the epidermis of Cameliria sativa. 

The asexual nonsporangial fruiting bodies of Pythium debaryanum 
are referred to as chlamydospores rather than as conidia, though in 
most of the previous literature the latter term has been used for 
them. Hesse called the terminal bodies conidia and the intercalary, 
gemmae (74). It is believed that the best terminology and the one 
which should be followed for all fungi, as it now is for most, is that 
which limits the term conidium to a spore which is adapted primarily 
for aerial distribution or which is at least readily separated as soon 
as it is mature from the parent hypha from which it arises. The 
most typical conidium, in fact, is a spore which is abstricted by the 
parent hypha at maturity. The asexual spores of this Pythium re- 
main attached to the parent hypha? indefinitely even after the hyphse 
are dead and empty. It is a common thing to find numbers of these 
bodies in water cultures, still attached to hyphse which are so com- 
pletely empty that it is only with favorable lighting that their thin 
colorless walls can be seen. So firm is the attachment that vigorous 
shaking is required to release any considerable proportion of the 
spores. It seems probable that in nature the spores are released 
chiefly as a result of the destruction of the hypha? walls by bacteria. 
While there is reason to think that Pythium debaryanum is some- 
times disseminated by wind, it is by no means certain that it is 
through the medium of these spores. It is true that these bodies have 
thinner walls than are commonly found in chlamydospores of some 
other fungi, but they have somewhat thickened walls as compared 
with the vegetative hyphse, and they are commonly intercalary. 
These facts, and (lie indications that they are better able to with- 
stand unfavorable conditions than are the hyphse, all tend to entitle 
them to rank as chlamydospores. De Bary (5) speaks of them as 
kv dauerconidia." Their ability to stand drying is not entirely demon- 
strated, but is indicated by the relative longevity of the fungus on 
different media. On beef agar and on rice, on which no spores are 



Bui, 934, U. S. Dept. of Agriculture. 



Plate I 




Pythium debaryanum from Artificial Cultures. 

Fig. 1.— Hyphae, showing old portions of hyph:« and false septa separating them from the portions still 
containing protoplasm. Figs. 2 to 4.— Various stages of oospore formation. Figs. 5 to 8.— Hyphal 
swellings at points of contact with glass. From camera-lucida drawings. 



DAMPING-OFF IX FOREST NURSERIES. 41 

formed, a few tests indicate that the fungus is very short lived, 
sometimes dying in a month. On media on which spores are pro- 
duced, transfers any time before the sixth month, and often as late 
as the tenth month, start immediate growth on fresh media. This 
is true even for strains which produce few or no oospores. The im- 
mediate commencement of growth from cultures 3 or 4 months old 
is taken as an indication that the new growth results from the 
asexual spores, as oospores are commonly believed to require a rest- 
ing period of five or more months before they are able to ger- 
minate (5, 38). 

INOCULATION ON STERILIZED SOIL. 

Pythiwn debaryarwm has been used in inoculation in pots of 
recently autoclaved soil in 16 different series of tests. In 10 of these, 
fragments of agar cultures were scattered over about one- fourth 
of the area at the side of each pot when seed was sown; in 2 of these 
10 and also in 2 other tests some pots were inoculated over their 
entire surface. In every one of these 12 heavily inoculated series 
positive results were indicated by smaller emergence and where any 
considerable number of sprouting seeds escaped the fungus by heavier 
damping-off loss in the inoculated pots than in the controls. In 
some cases the fungus killed all or practically all of the seed or 
seedlings in the inoculated pots before they emerged from the soil. 

In a total of 7 series, part or all of the pots received lighter in- 
oculations, consisting of one or two small fragments of an agar 
culture placed just below the surface of the soil at the edge of each 
pot. In 5 of these success was indicated. In the sixth and seventh 
also of these lightly inoculated sets, there was more damping-off in 
the inoculated pots than in the controls, but the difference was neg- 
ligible. The damping-off caused by light inoculations was in general 
distinctly less than that resulting from broadcast inoculations. To 
sum up the evidence : Sixteen separate experiments were conducted 
with Pythium debaryanum on pine seedlings in autoclaved soil, and 
in every one fewer seedlings survived in the inoculated pots than in 
the checks ; the difference in most of the experiments was large. 

Of the successful inoculation experiments — that is, those in which 
the difference between the inoculated pots and the checks seemed 
significant — 9 series included jack pine (Pinus banksiana) , 7 series 
western yellow pine (P. ponderosa, Colorado and New Mexico seed), 
and 3 series red pine (P. resinosa). In addition to the pines, Doug- 
las fir (Pseudotsuga taxifolia, Colorado seed) was grown in two large 
plats in one of the earlier series, one being inoculated over its entire 
surface with Pythium debaryanum. Because of the poor quality 
of the seed in the test on Douglas fir, too few seedlings were obtained 
to furnish a decisive test, but the difference in the emergence in the 
inoculated plat and the control affords preliminary evidence that 



42 



BULLETIN 934, U. s. DEPARTMENT OF A.GRICULT1 RE. 



Pythium can cause the "germination-loss" type of damping-off in 
Douglas fir as well as in specie's of Pinus. Of the seeds sown in the 
control plat !•"> produced seedlings which appeared above the soil, 
while only two seedlings appeared from an equal number of seeds 
sown in the inoculated plat. 

Altogether. 38 strains, excluding reisolations, have been tested on 
one or more of the 3 pine species. Strains from both the Pacific 
coast and the eastern United States and from a number of hosts 
other than pines were among those used. With the exception of two 
or three strains from a pine nursery in Michigan, the use of which 
was followed by so little damping-off as to leave their parasitism 
uncertain, all of the strains proved parasitic under favorable condi- 
tions, though some were more virulent than others. The positive re- 
sults in the 14 successful experiments are based on the comparison 
of a total of approximately 1,160 inoculated pots with 195 control 
pots. 



Table III. 



-Inoculation experiments with Pythium debaryawum in pots of steri- 
lized soil. 





Pythium strain. 


Num- 
ber 
of 

potS. 


Inoculation 
method. 


Results. 


Series, experiment number, 
and host. 


No. 


Initial st rain 
from which it 
wasreisolated. 


Emerged 

1 per 5-pOl 
unit ). 


Damp- 
ing-oil 

after 
germi- 
nation 

(per 
cent). 


Sur- 
vival 

( per .">- 

pot 

unit). 


Series A.— Initial inocula- 
tions: 


[218 




5 

5 
5 

5 
5 

5 

5 
5 
6 
5 

5 
5 
5 

5 
5 


Agarcultures 

broadcast 

atoneside 

of pot. 

Noinoculuni 

do 

Agarcultures 
at single 
point in 
each pot. 

Noinoculuni 

Agarcultures 

broadcast 
atoneside 
of pot. 

do 

Noinoculuni. 

do 

do 

Agarcultures 
broadcast 
atoneside 
of pot. 

do 

do 

No inoculum 

(>) 


1 

74 
82 

41 

55 

16 

18 
90 
82 
82 
14 

3 
13 

78 

14 

78 


100 




2S 



81 

89 




72 

67 

67 



72 






No. 5S, Pinus banksiana'. . 


Controls. 

..do 




74 






82 




[295 




30 


No.5S, Pinus ponderosa.. 






55 




[258 




3 




..do 




2 




Controls. 

do 




90 






82 




.do 




82 








4 




347« 




1 




348a 




4 




.Controla 
1338 

(controls. 




78 


SeeiesB.— Reisolatedstrains: 
>2, Pinus banksiana. . 


No. 295 (in P. 
ponderosa, 
expt. 58). 




4 

78 



«.V different soil used in these pots from that used wit h strain 25S and the first three control units. 

J>A11 inoculations with fragments of agar cultures scattered broadcast at one side of the pot, including 
about one-fourth of its area. Nothing was added to the controls in experiment 62, but sterile agar was 
added to the controls in experiments 66, 67, and 68. 



DAMPING-OFF IX FOBEST NURSERIES. 



43 



Table III. — Inoculation experiments with Pythium debaryanum in pots of steri 

lized soil < 'ontinued. 





Pyth 


nun train. 


Num- 






te nil . 












Damp- 




Series, experiment number, 
and host. 


No. 


Initialstrain 
from which it 


ber 

of 
pots. 


Inoculat inn 
method. 


Emerged 
i per 5-pol 


ing-oil 
after 
germi- 


Sur- 
vival 
(per 5- 






wasreisolated. 




unit i. 


nal ion 


pof 














(per 


unit i. 














cent). 




Series B.— Heisolated strains- 
















Continued. 


















338 


No. 295 (in P. 
ponderosa, 
expl . 58). 


5 


(a) 


9 


67 


3 




345 


No. 218 (in P. 
banksiana, 
expt. 58). 


5 


(a) 


15 


33 


10 




414 


No. 258 (in P. 

banksiana. 


5 


(a) 


36 


25 


27 


No. 06, Pinus banksiana. . 




expt. 62, 2d 

unit). 














415 


do 


5 


(a) 
(«) 


59 


10 


54 




419 


No. 348 (in P. 


5 


25 


100 









banksiana, 
















expt. 62). 














450 


No. 347 (in P. 
banksiana, 
expt. 62). 


5 


(") 


41 


72 


11 




( (Jilt nils. 
33S 




25 
5 




75 

7 


14 
57 


64 




No. 295 (in P. 


( a ) 


3 






ponderosa, 
















expt. 58). 
















No. 218 (in P. 

banksiana, 
expt. 58). 


5 


(a) 


24 


37 


15 




414 


No. 258 tin P. 

banksiana, 
expt. 62, 2d 

unit). 


5 


(a) 


57 


24 


44 


No. 67, Pinus banksiana. . 




No. 258 (in P. 
banksiana, 
expt. 62, 1st 

unit i. 


5 


(a) 


30 


37 


19 




419 


No. 348 (in P. 
banksiana, 
expt. 62). 


5 


(a) 


62 


65 


22 




450 


No. 347 (in P. 
banksiana, 
exnt. 62). 


5 


(a) 


53 


27 


39 




(.Controls 
338 




23 
5 




87 

85 


5 

26 


83 




No. 295 (in P. 


(a) 


63 






ponderosa, 
















expt. 58). 














345 


No. 218 (in P. 
banksiana, 

expt. 58). 


5 


(a) 


76 


24 


58 




414 


No. 258 (in P. 
banksiana. 
expt. 62, 2d 
unit). 


5 


(») 


98 


14 


84 


No. 68, Pinus resinosa 


415 


No. 2.W (in P. 
banksiana, 
expt. 62, 1st 

unit ). 


5 


(•) 


92 


11 


82 




419 


No. 348 (in P. 

banksiana, 
expt. 62). 


5 


(«) 


95 


45 


52 




4.50 


No. 347 (in P. 
banksiana, 
expt. 62). 


5 


(o) 


84 


40 


51 




[Controls 




18 




104 





104 











a All inoculations with fragments of agar cultures scattered broadcast at one side of the pot, including 
about one-fourth of its area. Nothing was added to the controls in experiment 62, but sterile agar was 
added to the controls in experiments 66, 67, and 68. 

As has been stated, securing positive results did not always mean 
that the control pots remain uninfected. Even with the most care- 
ful treatment and the use of boiled water throughout the experiment 



14 BULLETIN 934, r. S. DEPARTMENT OF AGRICULTURE. 

it proved difficult to keep the control pots entirely free from damp- 
ing-off. Cultures from seedlings which damped-off spontaneously 

iu control pots indicated that Pythium us well as Fusarium may be 
introduced by accident, ever when insects, birds, and rodents are ex- 
cluded. This agrees with the evidence of Hofmann (77) that 
Pythium debanjanum is sometimes disseminated by wind, despite 
its apparent lack of adaptation to wind distribution. It is also in- 
dicated, however, that unheated tap water increases damping-off 
when used on control pots and probably carries this semiaquatic 
fungus. Notwithstanding infections in the controls of a number of 
the experiments, it is believed that the large number of pots whose 
results have been considered in drawing conclusions, the fact that the 
Pythium pots lost more heavily than the controls in every one of the 
16 experiments, and the magnitude of the differences between both the 
emergence and subsequent damping-ofF figures for the inoculated pots 
and the controls in most of the experiments establish the parasitism 
of the fungus in inoculation on autoclaved soil without it being neces- 
sary to present all the evidence in detail. The pot series which in- 
volved reisolation and reinoculation (Table III), together with the 
results given for other purposes in Tables V and VI, seem sufficient 
by themselves to establish a parasitic relationship. 

REISOLATION AND REINOCULATION. 

In a number of the experiments dead seedlings in the inoculated 
pots were examined and typical Pythium hyphse and spores were 
found. In three of the experiments in which the controls remained 
entirely free from disease up to the time the experiment was closed, 
reisolations and reinoculations were made in accordance with the 
usual rules of proof. The results are given in Table III. 

From Table III it will be seen that five strains reisolated from 
Pinus banksiana and one strain reisolated from P. ponderosa gave 
positive results in pots of P. banksiana and P. resinosa. In addition 
to the reinoculations shown in the table, the strain reisolated from 
Pinus ponderosa (No. 338) was again reisolated in duplicate from 
P. banksiana in experiment G2, and both these secondary reisolations 
gave cultures which w T ere parasitic on P. banksiana and P. resinosa 
in subsequent inoculations. 

That the organisms reisolated were actually the same as those used 
in the initial inoculation is indicated not only by the absence of dis- 
ease in the control pots of experiments 58 and 62, but by the distinc- 
tive characters of some of the strains. In general, cultures reisolated 
from strongly parasitic initial strains were themselves strongly 
parasitic and vice versa. This is shown by comparing the figures for 
the initial and reisolated strains, as shown in Table IV. Each figure 
represents the average results in 10 pots of jack pine and 5 of red 



DAMPING-OFF IX FOREST NURSERIES. 



45 



pine in experiments <'»<;, 67, and 68 combined. The figures are rel- 
ative* the mean survival of 47 different strains used in all three ex- 
periments being taken as 10. A survival figure above 1 ( » therefore 
means that the strain was less destructive than the average Pythium, 
and a figure below 10 indicates more than average virulence. As 
strain 218 was not used in these three experiments, strain 345 can not 
be compared. 

Table IV. — Comparative virulence of original cultures and reisolated strains of 
Pythium dehunjunum in c.vperhuciit.s (Hi, 67, a in! 68. 



Pythium strain. 


Description. 


Rela- 
tive 

sur- 
vival. 


Pythium strain. 


Description. 


Rela- 
l i\ r 
sur- 
vival. 


No. 258 


i Original culture 


16 
12 
12 
4 
4 
6 


No 409 


Reisolated from 338 




No. 414 


Reisolated from 258 

do 


No. 347... 




No. 415 


No. 450 


Reisolated from 347 


7 
10 
4 


No. 295 


< >riginal culture 


No. 348 


No. 338 


Reisolated from 338 


No. 419 


Reisolated from 34S 


No. 408 









These figures are not absolutely consistent, but are to be viewed 
as contributing to the evidence furnished by the absence of damping- 
off in the control of experiments 58 and 62 that the cultures reiso- 
lated in those experiments were actually identical with the original 
strains. A further proof of this identity is in the fruiting tendencies 
of the strains. Both Nos. 414 and 415, the strains reisolated from 
original strain 258, exhibited the peculiarly sparse spore production 
which has been characteristic of strain 258 for the entire period dur- 
ing which it has teen in culture. The other reisolated strains, taken 
from pots inoculated with normally fruiting strains, all showed 
normal spore production. 



PURITY OF CULTURES. 



A slight deficiency in the evidence as to the parasitism of Pythium 
debaryanum both in the writer's work and apparently in all previous 
investigations except those of Peters (100) and possibly Knechtel 5 
is the lack of single-spore cultures. The large number of strains 
which have remained apparently pure through numerous subcultures 
and have retained their individual characteristics as to virulence and 
fruiting tendencies (one strain having been carried on artificial 
media continuously for eight years without material change) give 
very strong justification for believing that the cultures used were 
pure. In three early inoculation tests the cultures used were after- 
wards found to have been contaminated *by bacteria carried by mites; 
the positive results obtained in these three were the basis of the ear- 

5 Knechtel's work in Rumanian has been available to the writer only in the German 
abstract, which makes an ambiguous statement on this point. 



46 BULLETIN 984, U. S. DEPARTMENT OF AGRICULTURE. 

liest report of pathogenicity (02), but have not been used as evidence 

in the present bulletin, though the contaminating bacteria in one of 
them, when tested independently, showed no evidence of parasitism. 
In all the experiments mentioned in the foregoing as giving positive 
results with Pythium the cultures used were apparently pure. 

Cultures from single chlamydospores should be reasonably easy 
to secure, part of the chlamydospores in water cultures being separa- 
ble from the mycelium by vigorous slinking, and further inoculation 
tests with cultures so obtained are probably desirable. The experi- 
ments so far conducted are believed to be sufficiently conclusive, how- 
ever, for all practical purposes. For isolation of absolutely pure 
lines of this or any other coenocytic fungus, it is evident, as pointed 
out by Dr. W. H. Weston (146), that isolations should be made from 
the uninucleate swarm spores. For the determination of the bare 
fact of pathogenicity such a refinement would be superfluous. 

CROSS-INOCULATIONS. 

The physiological identity of the Pythium attacking coniferous 
seedlings with the one which attacks dicotyledons is indicated by 
the results of several inoculation experiments. The last two experi- 
ments, one with jack pine and one with red pine for the host, are 
the most comprehensive and give results sufficiently decisive so that 
quotation of the corroborative evidence from earlier experiments 
is considered unnecessar}^. The results appear in Table V. Each 
unit consisted of five 3-inch pots except in the controls, in which 
23 pots were used in the jack-pine experiment and 18 in that with 
red pine. In the second experiment, separate records were kept of 
the survival in each pot, and the probable error calculated from the 
controls was less than two seedlings per pot for a single pot, less 
than 0.9 for a mean of 5 pots, and less than 0.5 for the mean of the 
18 control pots. While the number of controls was, of course, in- 
sufficient to furnish an exact basis for such a calculation, the small 
value found tends to confirm the impression gained from inspection 
of the table that considerable confidence can be placed in the results. 

The difference appearing in Table V between jack pine and red 
pine in point of susceptibility to germination loss from Pythium 
agrees with field observations in Nebraska, the red pine at the Bessey 
Nursery, though on the whole more susceptible than jack pine to 
dam ping-off losses, having given indication of more resistance to the 
disease for the first week or two. Inoculations in other experiments 
on western yellow pine indicate that the strains which attack it are 
identical \\ it li those attacking jack pine and red pine. 

The conclusion reached from the cross-inoculation results is that 
the Pythium causing damping-off of the three species of pine men- 



DAMIMNi; ul-'l IN L'ORKST N IT.KERIKS. 



47 



tinned is identical with PytMum debaryanvm, causing leak of potato 
tubers and the damping-off of seedlings of two dicotyledonous 

families. 

Table V. — Results j>( inoculations on jack pine and red pine with Pythium 
deoaryanum from various hosts. 





Host from which isolated. 


Inoculation results. 




On jack pine. 


On red pine. 




Emerged 

( per 5-pot 

unit). 


Damp- 

ing-oir 
(per 
cent). 


Sur- 
vival 
(per 
5-p ot 

unit). 


Emerged 
(per 5-pot 

unit). 


Damp- 
ing-off 
(per 
cent). 


Sur- 
vival 
(per 
pot). 


No. 131 a 


Dicotyledons: 


45 
45 


36 
34 


30 
30 


101 
62 


23 

11 


15.6 


No 8106 


Do 


7.4 










45 


35 


30 


82 


32 


11.6 








No. 294 c 


50 

28 
19 


8 

32 
32 


46 

19 
13 


79 

62 
68 


36 

48 

58 


10.2 


No. 295 c 


Originally potato strain 131, but 

1 wire inoculated on and reisolatod 
from sugar-beet seedlings by Ed- 


6.6 


No. 296d 




5.8 










32 


24 


26 


70 


47 


7.4 








No. 529 


36 1 31 

60 49 


25 
31 


102 
108 


26 
22 


15.0 


No. 530' 


Do 


16.8 




4.8 40 


28 


105 


24 


16.0 




Conifers: 

Western yellow-pine seedlings 




No. 258 


58 


9 


53 


109 


17 


18.0 


No. 550 


15 1 80 
42 29 


3 

30 


39 
45 


-IS 

70 


.2 


No. 555 




5.0 




Average, spruces 

Controls 






29 ' 55 


17 


42 


84 


2.6 




87 


5 


83 


104 





20.9 



n Furnished by Mrs. C. R. Tillotson- has been used b Furnished by Dr. L. A. Hawkins: cause of leak, 
successfully on sugar-beet seedlings by Dr. H. A. c Furnished by Dr. H. A. Edson. 
Edson. d Diseased material furnished by Prof. W. T. Home. 



VARIATIONS IN VIRULENCE OF PYTHIUM STRAINS ON PINE. 

In Pythium debaryanum strains, as in the case of Corticium 
vagum, there appeared to be a considerable difference in the parasitic 
activity of different strains used in the same experiment. Figures 
14, 15, and 16 show graphically the results from inoculations with 
different strains of P. debaryanum in all the experiments in which 
it was possible to compare directly the activity of different strains. 
All the inoculations involved at the time of sowing the addition to 
the soil of cultures on nutrient media in recently autoclaved 3-inch 
pots. In experiment 31C the inoculum fragments were scattered over 
the whole pot, in 31D at only one point in each pot, and in the others 
were distributed over about one-fourth of the pot's area. As noted 
elsewhere, the variations observed in the results may have been due 
in part to differences in the ability of the different strains to main- 



48 



BULLETIN 934, l T . S. DEPARTMENT OF AGRICULTURE. 



tain themselves saprophytically in the soil used rather than entirely 

due to difference in virulence. 

The data shown in figures 15 and 1G indicate in the first place 
rather more accidental variations in the results with Pythium than 
with Corticiuin (see figs. 1, 2, 10, and 11). The agreement between 
original and reisolated strains from the same original source is de- 
cidedly less good than in the case of Corticium (see experiments 71 
and 72, figs. 10 and 11). In general, there are only two strains of 



HOST 
Y£/>/? 
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Fig. 14. Diagram showing variations in virulence as indicated by the living seedlings 
in pots of autoclaved soil inoculated with different strains of Pythium debaryemum. For 
experiments Nos. .'{1 and 66 to 72, inclusive, the surviving seedlings at the end of two 
or three weeks after germination are shown. For the other experiments daniping-off 
was so heavy in the inoculated pots that the survivals did not give differential results 
for the different strains, and the germinations are therefore shown. The reports are 
based for experiments Nos. 71 and 72 on 2 or 3 pots for each strain in each experiment, 
and for the other experiments on not less than 5 pots for each strain. In experiments 
Nos. (!G, 67, and <iS the number of pots in each experiment for the strains whose 
reisolations were also used varied from 10 to 40 for each strain, the results of the 
separate 5-pot units being shown in figure 15. The strains indicated by the different 
symbols are as follows: From potato: ©=Strain 131, isolated in 1909, California. 
Furnished by Mrs. C. K. Tillotsou. From sugar beet: <0=Strain 205 and its reisola- 
tions from pine. No. 295 was furnished by Dr. H. A. Edson as a reisolation of strain 
131, after having been passed by him through two generations of sugar-beet seedlings. 
▲ =Strain 294, isolated in 1912. Furnished by Dr. Edson. A=Strain 296, isolated 
in 1912, Wisconsin. Furnished. by Dr. Edson. X=Strain 297, originally from pine, 
Nebraska, 1911. Passed through two generations of sugar-beet seedlings by Dr. Edson. 
From pine .seedlings: +=Strain 255, Kansas, 1913. Chlamydospores numerous ; oospores 
rare. H=Strain 258 and its reisolations, Kansas, 1913. A sparsely fruiting strain. 
□ =Strain 218 and its reisolation, Kansas, 1912. ©=Strain 347 and its reisolation, 
Washington, D. C, 1915. H=Strain 348 and its reisolation, Washington, D. C, 
1915. A=Strain 349, Washington, D. C. 1915. H = Strain 354, Minnesota, 1915. 

Pythium which can be said to have definitely shown difference in 
activity continuing through several years and on different species of 
pine. These are strains 295 and 258. As No. 258, the weak strain, 
has also been found abnormal in its fruiting tendencies, the evidence 
in these graphs does not indicate a decided difference in virulence 
between different typical strains of Pythium debaryarwm. The 
other strain, which seems rather uniformly weaker than No. 295, is 
No. L31, which according to Dr. Edsoifs records was originally the 



DAMPING-OFF IX FOREST NURSERIES. 



49 



same strain, No. L31 having been twice used in bis inoculation ex- 
periments on sugar beets and strain 295 recovered from the second 
experiment. The apparent difference between this original strain 

and its supposed reisolation may possibly be due to the treatment 
given strain 131. Before it was used in any of the experiments 
shown but after it had been used by Dr. Edson, it was allowed to 
get very dry and was revived with great difficulty, growth being 
very slow. While it apparently recovered all of its normal growth 
qualities after one or two transfers, it is thought that this may 
possibly explain the 
apparently decreased 
virulence in the later 
experiments. 

The failure to se- 
cure as definite indi- 
cations of constant 
virulence differences 
as were obtained for 
several of the Cor- 
ticium strains is be- 
lieved to be in part 
due to a smaller 
actual difference be- 
tween the different 
Pythium strains ap- 
pearing in the graphs 
and in part to a 
larger accidental va- 
riation between re- 
sults in pots inocu- 
lated with the same strain. The growth of Pythium on agar media is 
much more affected by variations in the substratum than is the growth 
of Corticium, and it is rather natural to expect greater variations when 
the two fungi are added to autocla ved soil. In experiments 66, GT, and 
68 a number of strains not used in the earlier experiments were tested, 
in addition to the strains previously used. The survival results for 
all the different strains, both original and reisolated, 47 in all, are 
shown graphically in figure 16. The results in experiments 66 and 
67, both on Pinus banksiana, are averaged and taken as the subject, 
while the results with the same strains in experiment 68 are made 
relative and shown by the broken line. The correlation between the 
performance of the same strains on the two species of pine is by 
no means as clear in the graph as it was in the case of the Cor- 
ticium strains (fig. 11). The areas bounded by the broken line and 
19653 -Bull. 934—21 4 



/>//Vt/S &#NK&/#N# 


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67 


63 


m 

: 

HP E3£IG> °OC\ 


■ 

* 

40 



hosa 

YEAR 
EXPT. 

||* 

Ete 

Fig. 15. — Diagram showing the results of inoculations with 
strains of Pythium debaryanum. This figure supplements 
figure 14, giving the results for original and reisolated 
strains independently. Each point plotted is base. I on 
the results in five pots. The object of this diagram is to 
give an idea of the degree of variability in the success of 
inoculations. An explanation of the symbols used will 
be found in the legend of figure 14. 



50 



BULLETIN 934, U. S. DKI'AKTMKNT OK AC.KIGXJLTURE. 



the horizontal line showing the locution of the mean for experiment 
68 are much larger below the moans than above it in the left-hand 
portion of the graph, while the reverse is true in the right-hand 
portion. To this extent the relative activity of the strains in this 
experiment agrees with the performance of the same strains in the 
two jack-pine experiments, as shown by the solid line. It can not 
be decided from an inspection of the graph whether there is a real 
agreement, in view of the large accidental variation present. How- 
ever, the correlation coefficient, 0.446+ 0.079, five and one-half times 
its probable error, indicates a considerable correlation, not as good 
as was found for the Cbrticium strains, but sufficient to establish a 
strong presumption that observed differences in activity of the dif- 



i 

I 



A 



/•% 




I A / S / i 

1 J \ j^AYJrJff- 



M£#/V eXPTS. 66/7/VO 67 



ill' I L_l l_ 



'I'll' 









m\ 



Fig. 16. — Diagram showing the comparative virulence of 47 strains of Pythium debar yanum 

in successive inoculation experiments on species of Tinus. The results in experiments 
Nos. 66 and 67 (on Pinus banksiana) are shown by the solid line, the strains beinj; 
arranged from left to right in the order of descending virulence indicated by the number 
of seedlings surviving in those experiments. The results from the use of the same 
strains in experiment No. 68 (on Pinus resinosa) are shown by the broken line. Such 
correlation as there is between the two curves (coefficient 0.45±0.08) goes to indicate 
a real difference in virulence between the different strains. The strains indicated by 
(he underscored numbers are original strains, -and those not underscored are reisolations 
from the original strains in earlier inoculation experiments on pine seedlings. 

ferent strains in these inoculation experiments were in part actually 
due to differences in the capacity of the strains. 

It has been suggested in the foregoing that the difficulty in demon- 
strating constancy in the difference in virulence between the various 
strains of Pythium debaryanum is due in part to the lack of such 
extreme differences as were observed between the various Corticium 
strains. Figure 13 shows the distribution of the different original 
Pythium strains according to the virulence indicated in the three 
inoculation experiments of figure 16 (application to autoclaved soil 
at the time of sowing). Each value plotted is based on the average 
results in 15 pots. Of the strains used, 21 were from species of pine, 
1 from spruce, 2 from potato tubers, 2 from fenugreek, 3 from sugar 
beet, and 6 from soil direct. Despite the considerable number of 



DAMPING-OFF IN FOREST NURSERIES. 51 

strains, they are not much more representative than the smaller num- 
ber of Cortieium strains experimented with. All of the strains from 
soil direct and 11 of the strains from pine were taken at approxi- 
mately the same time from the same nursery in Michigan hy Mr. 
(Jlenn G. Halm; despite the fact that these were the most recently 
isolated of the strains used, nearly all of them proved weak in the 
inoculations. Of the 17 strains which proved the weakest (out of 
35), all but 3 were from this Michigan nursery. The 18 strains from 
other sources (5 from California, 2 from Minnesota, 2 from Kansas, 
1 from Wisconsin, 2 from an unknown locality, and 6 from Washing- 
ton, D. C, representing two coniferous and three dicotyledonous host 
genera), as shown by solid circles in figure 13, for the most part were 
rather closely grouped within the more virulent portion of the range. 
The coefficient of variability in the survivals allowed by the 35 
Pythium debaryanum strains is 39±3.6 per cent, while for the 
smaller number of original strains of Cortieium vagum in experi- 
ments 71 and 72 it is 63 ±9.7 per cent. It is evident from figure 13 
that if there had not been a disproportionately larger number of 
strains from the Michigan nursery the variability of the P. de- 
baryanum strains would have been much less than 39 per cent. 
The number of strains was, of course, altogether insufficient for either 
fungus to represent adequately a population as immense as the total 
number of strains of either of these omnipresent species. The above 
data, however, contain the only available information of which the 
writer is aware on variation in the virulence of different strains of 
P. debaryanum. 

The evidence as a whole, both from the results shown in figure 13 
and the experience with 6 other strains Avhich were not used in the 
experiments on which figure 13 was based, lead the w r riter to believe 
that most strains of Pythium debaryanum taken from lesions in 
plants are ordinarily likely to prove rather virulent parasites on pine 
seedlings. It further appears that the variation in virulence between 
the different strains of P. debaryanum on pine seedlings is less than 
the variation in strains of Cortieium vagum. 

PYTHIUM INOCULATIONS ON TJNHEATED SOIL. 

Inoculations with Pythium debaryanum were made in western 
Kansas on a fine sand containing little humus after treating the soil 
with acid followed by lime. Commercial sulphuric acid was applied at 
the rate of 14.8 c. c. per square foot of bed, followed two days later 
by 25.5 grams of air-slaked lime raked into the soil (0.16 liter of 
acid and 0.274 kg. of lime per square meter). The acid was diluted 
before applying with 256 volumes of water. The seeds were sown 
in drills, and inoculum was placed in the drills at the time of sowing. 
Each unit involved approximately 11 linear inches of drill, and all 



52 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

received equal quantities of seed. Three strongly parasitic strains 
of Pythium were used, and a total of 12 units of jack pine and an 
equal number of western yellow pine was inoculated with 12 inter- 
spersed units of each species as controls. The mean results arc as 
follows : 

Pirms banksiana. — Inoculated plats: Emerged, 64.2±4.9; died during the next 
17 days. 25 per cent. Control plats: Emerged, 85.5±3.6; died during 
the next 17 days, 13 per cent. 

Pinus ponderosa. Inoculated plats: Emerged, 34.6±1.8; died during the aexl 

9 days, 39 per cent. Control plats: Emerged, 45.4±1.3; died during the 
next 9 days, 25 per cent. 

The difference in emergence apparently due to the inoculation is 
for the first species three and one-half and for the second nearly five 
times its probable error. While, of course, 12-unit means are in- 
sufficient to allow the calculation of entirely reliable probable errors, 
they give some idea of the amount of variability of the results and 
the confidence which can be given them. It is impossible to give any 
such expression applying directly to the damping-off percentages and 
their differences, for the reason that averages for this item have 
been made in the writer's work not by averaging the percentages for 
the individual units but by totaling all the seedlings and the dead 
seedlings on the plats to be averaged and recalculating the percent- 
age from these figures. This seems the only safe method, as other- 
wise units in which germination is low by accident or by the action 
of parasites will be given an influence on the resultant mean entirely 
disproportionate to the number of seedlings which they contain. 
Average values for damping-off obtained by this method and by the 
method of averaging the percentages of the individual plats or pots 
are often very different; it not uncommonly happens that the units 
in which germination is lower than the average also have especially 
high damping-off percentages, both phenomena being caused by an 
unusual activity of parasites. In such case to average the percentages 
themselves usually gives a higher damping-off figure than to total 
the seedlings for the different units and redetermine the percentage, 
and the latter practice is considered the better. In the present case 
the differences in the damping-off percentages obtained by the two 
methods are not great. The figures obtained by averaging the per- 
centages of the ultimate units are as follows: 

Pinus banksiana. — Inoculated, loss 30.9±5.0 per cent; controls, loss 13.2±2.S 

pel- cent. 

Pinus ponderosa.— Inoculated, loss 40.0±5J per cent; controls, loss 24.1±:$.3 
per <en I. 

The differences between the inoculated and control plats in damp- 
ing-off percentage were for the first species a little over and for the 
second a little under three times their indicated probable errors. 



DAMPTNG-OFF IX FOREST NURSERIES. 53 

The results in general make it appear (hat the Pythium was able to 
kill some pines both before and alter their appearance above the soil 
surface on the soil treated with the acid and lime. The control in 
this experiment did not receive the nutrient substratum added with 
the Pythium inoculum, but an experiment run under the same con- 
ditions at nearly the same time, in which seven strains of hyphomy- 
cetes with the same substrata entirely failed to decrease survival, 
indicates that the rice subtratum was not in itself the cause of the 
observed result. The rather weak action of the Pj'thium in these 
experiments stands out in sharp contrast to the results with Corticium 
vagum in the same experiments, practically all emergence being pre- 
vented by most of the Corticium strains used, some of which had 
proved less active than Pythium in tests on autoclavecl soil. 

In a soil in Nebraska, somewhat similar but with more humus, 5.5 
c. c. (three-sixteenths fluid ounce) of sulphuric acid per square foot 
applied in solution at the time of sowing had been found greatly to 
decrease damping-off. In different parts of beds treated with acid 
from 10 to 17 days earlier, 96 plats, each 3 inches square, were laid 
out, and each plat was inoculated at the center. Interspersed with 
these were 96 plats set apart as controls. Emergence had already 
begun at the time of inoculation. Jack pine, red pine, and Gorsican 
pine were the hosts, and three Pythium strains of known parasitism, 
growing in pieces of prune agar the size of peas, constituted the 
inoculum. The damping-off after emergence was less than 1 per 
cent higher for the inoculated plats than for the controls. Even such 
a light inoculation would probably have given some results in auto- 
claved soil, so the experiment indicates, as would be expected, that 
this acid-treated soil was less favorable for Pythium debaiyanum 
than steamed soil. 

On pots containing entirely untreated soil the following series of 
inoculations were made at the time of sowing the seed : 

Inoculation at one point in each pot : 

Experiment 25. Jack and western yellow pine, 1 pot of each inoculated; 
survival 13 days alter emergence slightly greater in both than in the 
six controls. 

Experiment 27. Jack pine, 73 pots, 27 controls; average emergence, 59 
in inoculated pots and HO in controls; damping-off, 39 per cent in 
inoculated pots and 37 per cent in controls. 

Experiment 29. Jack, Corsican, and western yellow pine, 112 plats in- 
oculated just as emergence commenced instead of at seed sowing, 
as in other cases, and 112 controls alternating with them ; damping- 
off was less in the inoculated plats than in the controls. 

Experiment 31. Jack pine, 8 pots inoculated, 8 controls ; inoculated, 
emergence 33 per cent, damping-off 13 per cent, survival 198 ; con- 
trols, emergence 38 per cent, damping-off 26 per cent, survival 196. 

Experiment 58A. Jack pine, 5 pots inoculated, 5 controls; inoculated, 
emergence 59 per cent, damping-off 32 per cent, survival 40; controls, 
emergence 51 per cent, damping-off 12 per cent, survival 45. 



54 BULLETIN 934, 1 T . S. DEPARTMENT OF AGRICULTURE. 

Inoculations :it two points In ea< b pol : 

Experiment 26A. .lack pine, .*? pots Inoculated, 4 controls; Inoculated, 
emergence '-".> per cent, as compared with •'!!' per cent in the controls; 
subsequent damping-off the same in both. 
Inoculations al tour points in each pot: 

Experiment 58B. .hick pine, 5 pots inoculated, 5 controls; inoculated, 
emergence 51 per cent, damping-ofE 10 per cent, survival 46; controls, 
emergence 48 per cent, damping-off 22 per cent, survival 84. 

Experiment 59A. Jack pine, 5 pots inoculated, 5 controls; inoculated, 
emergence 55 per cent, damping-ofl 2 per cent, survival 54; controls, 
emergence 50 per cent, damping-ofE S per cent, survival 4C. 

Of these experiments, No. 29 was in the original fine sandy soil 
of a nursery in Nebraska in which Pythium is commonly found 
native and damping-off losses are usually heavy. Experiments 58A 
and 59 were conducted on soil from the same source which had, been 
kept dry in the laboratory for five years; experiments 25, 27, 31, 
and 58A were on greenhouse mixtures of sand and soil. In experi- 
ments 31, 58 A, 58B, and 59 parallel inoculations were made on auto- 
claved portions of the same soil, with definitely positive results in 
three of the four cases. In the heated soil the results were positive. 
not only because of smaller losses in the controls but because the losses 
in the inoculated pots were actually heavier in the sterilized soil than 
in that untreated. 

Inoculations broadcast : 

Experiment 31. Jack pine, 8 pots inoculated, S controls; inoculated, 
emergence 31 per cent, damping-ofE 39 per cent, survival 129; con- 
trols, emergence 3S per cent, damping-ofE 26 per cent, survival 196. 
Experiment 59. Jack pine, 5 pots inoculated, 5 controls; inoculated, 
emergence 58 per cent, damping-ofE 22 per cent, survival 45 ; controls, 
emergence 44 per cent, damping-ofE 2 per cent, survival 43. 

Even with these broadcast inoculations the results on untreated 
soil were too indefinite to allow the drawing of positive conclusions. 
In both experiments much heavier losses than these resulted from 
inoculations on steamed soil. It is evident that experiments on steril- 
ized soil do not always show what can be expected on ordinary soil. 
The same tiling is indicated by the results of Edgerton with tomato 
wilt (36). 

CONCLVSIONS AS TO THE PARASITISM OF PYTHIUM DEBARYANUM. 

Pythium debarijanum has been found in low-altitude nurseries in 
all the species of conifers from which a serious effort has been made 
to obtain it, and its parasitism has been indicated in autoclaved soil 
on all of the conifers on which inoculation has been attempted. 
Therefore, although the work reported has been limited to a relatively 
small number of hosts, it seems likely that it will be found able to 
cause damping-off in most of the species of the Abietoidese which 
suffer seriously from the disease. Just how active as a parasite it is 



DAMPING-OFF IX FOREST NURSERIES. 55 

under ordinary nursery conditions is yet to be proved. The results 
in inoculations on disinfected soil, together with the frequency with 
which the fungus has been isolated from seedlings in the nurseries, 
lead the writer to believe that it is an important cause of disease in 
the seed beds. Further experiments on unheated soil, however, are 
considered desirable. 

RHEOSPORANGIUM APHANIDERMATUS. 
CULTUBAL STRAINS. 

A culture of a parasite on radishes and sugar beets, described by 
Edson (39) under the above name, was obtained from him, and an- 
other strain, shown by Edson's records to be a subculture from the 
same original strain, was furnished by the department of plant pathol- 
ogy of the University of Wisconsin. In parallel cultures on solid 
media this fungus proved in many ways remarkably like Pythium 
deharyanum, reacting in practically the same way to the different 
media on which it was grown both in relative growth rate and in 
spore production. Mycelium, chlamydospores, oogones, antheridia, 
and oospores are not recognizably different from those of Pythium 
deharyanum. The oospores have seemed on the whole slightl} 7 larger 
and the mycelium a little more inclined to aerial growth than most 
of the Pythium deharyanum strains, but neither difference was suffi- 
cient to have diagnostic value. Swellings of the hyphse occurred at 
points in contact with glass, just as with Pythium deharyanum (PL I, 
figs. 5 to 7). 

In liquid cultures the Rheosporangium was readily distinguished 
from Pythium by the formation of the presporangia described by 
Edson. Autoclaved cylinders of turnip, 15 to 20 mm. long, cut with 
a 5-mm. cork borer, proved convenient bases for growth of both 
Rheosporangium and Pythium in water culture and quite as satis- 
factory as sterilized beet seedlings. Presporangia were also pro- 
duced in autoclaved soil, and in u single lot of corn-meal agar they 
were formed abundantly in the agar in Petri dish cultures. In none 
of the writer's cultures, either with flies, sugar-beet seedlings, or 
turnip cylinders as nutrient bases, were mature escaped sporangia 
or swarm spores commonly produced. 

The Rheosporangium was not obtained in any of the numerous 
cultures made from coniferous seedlings or from seed-bed soil. 

INOCULATION EXPERIMENTS. 

The Rheosporangium cultures above referred to, strain 229 fur- 
nished by Dr. Edson and strain 351 received from the University of 
Wisconsin, were tested on pine and red-beet seedlings, with parallel 
inoculations with Pythium deharyanum. The results appear in 
Table VI. 



56 



BULLETIN 934, CJ. S. DEPARTMENT OF AORTrrT/TURE. 



Tabi i VI.- Results of parallel inoculation with Rheosporangium aphanidermatut 
mid Pythium debaryanum on pine seedlings in autociaved soil. 



Experiment number, host, and inoculating fungus.' 



No, 30, Pinus ponderosa: 

Rheosporangium, strain 229. - 

Pythium, 2 strains 

Controls 

No. 31, Pinus banksiana: 

Rheosporangium, strain 229 

Pythium, strain 225 

Controls 

No. 58, Pinus banksiana: 

Rheosporangium, strain 229 

Pythium, 8 strains 

Controls 

No. 59, Pinus banksiana : 

Rheosporangium, strain 229 

Pythium, 8 strains 

('nil Iri iN 

No. 61, Pinus banksiana: 

Rheosporangium, strain 35] 

Pythium, 2 strains 

Controls 

No. 61, Funis banksiana and beets in same pots: 

Beet? [Rheosporangium, strain :i5l 

No. 61, beets alone: 

Rheosporangium, strain 351 

No. 62A, beets: 

Rheospora„gium{^;n^. ■-...-• --...-...- 

Pythium, 2 st rains 

Controls 

No. 62A, beets: 

Rheosporangium {^^ ;:;;;;:;:;:::;;;:: 

Controls 

No. 62A, beete and Pinus banksiana in same pots: 

g™^>Rheosporangium, strain 229 

gj^?| /Rheosporangium, strain :i">i 

Be»ontrols 

No. 62B, Jack pine: d 

Rl,e,,spo,,n,i,,mgraiu2|9... ................ 

Pythium, strain 258 

Controls 

No. 66, Jack pine: 

Rheosporangium^-;;;^;;;;;;;;;;;;;;;;; 

Pythium, 17 strains and substrains 

Cunt nils 

No. 67, Jack pine: 

Rbeosporangium^i?".-.:;;;;;;;;;;;;;;;; 

Pythium, 17 strains and substrains 

Controls 

No. 68, Red pine: 

Rheosporangium^ I?-;;;;;;;;;;;;;;;;;; 

Pythium, 17 strains and substrains 

Controls 



Number 

ol pots. 



235 
25 

5 

5 

235 

23 



5 
23S 

18 



Result; 



Emerged Damping- 
i per cent ofl i |>er 
ofseed). vent). 



/'< r pot. 
23 

II 
20 

45 

12 

43 

Per 5-pot 

unit. 

46 

2.6 



89 

12 
12 
86 

50 
c27 
c77 

r,l 
42 
46 
27 
81 
67 

58 
53 

10 
82 

63 

80 
43 



107 
ss 
51 
87 

105 

124 
86 

nil 



Per pot. 
3 



(*) 



88 
72 

1 

:« 
83 


20 

78 
1 

14 
22 
33 
78 



14 

10 

100 





a Location of the inoculum: In experiment 30, at one point at the edge of each pot: in experiment 31, 
over the entire pot; in all other experiments, over one-quarter the area of each pot. 

b The breakage of the one seedling not killed while sprouting prevented the determination of results. 

c Double seed density in these pots: emergence and survival figures halved to allow direct comparison 
with other units. This high seed density may explain in part the higher loss in strain 351 than in strain 229. 

d Experiment 62B was conducted at the same time as 62A, but with a different soil. 

Table VI shows that in experiments 30 and 67 the loss was less in 
the Rheosporangium pots than in the controls and that in experiment 
68 the results were entirely negative, while in the remaining seven 



DAMPINO-OFF TN FOREST NURSERIES. f)7 

experiments the losses were heavier in the Rheosporangium puts. 
Especially in experiments <>l and 62A the evidence indicates very 
strongly that both germination loss and subsequent damping-off of 
the seedlings which come up can be caused by inoculation with Rheo- 
sporangium on jack pine under favorable inoculation conditions. It 
is, however, obvious that in all of the experiments the parallel in- 
oculations with Pythium debaryamMi gave much more positive re- 
sults. The Pythium was active under conditions in which the Rheo- 
sporangium gave no evidence whatever of parasitic capacity. It 
furthermore appears that the two strains of Rheosporangium, though 
probably identical originally, differed in virulence at the time of 
their comparison in these experiments. The greater virulence of 
strain 351 was quite distinct in most of the comparative tests on beets 
as well as on pines. The possibility that the original culture was 
really a composite of two or more strains, of which different ones 
survived in the subcultures kept at Washington and Madison, re- 
spectively, seems worth considering. Such an accident might also 
have been responsible for the divergence of Pythium strains 131 and 
295 referred to in another section. 

Further evidence of the parasitism of Rheosporangium was ob- 
tained in inoculations with cultures reisolated from seedlings killed 
by the original strains in experiment 62. Typical Rheosporangium, 
identified by presporangium formation, was easily recovered from 
the damped-off seedlings in pots of pines only, those of beets only, 
and the pots in which both hosts were sown. The recovery of a 
virulent Pythium strain from a single one of the pots inoculated with 
the weaker Rheosporangium shows that despite the absence of dis- 
ease in the controls a slight amount of contamination did occur. 
However, the comparative ease w^ith which the Rheosporangium was 
isolated from seedlings in other pots inoculated with it and the fact 
that it has never been obtained in the numerous cultures made from 
controls and from pots inoculated with other organisms leave little 
room for doubt that the strains isolated were really recoveries of the 
Rheosporangium used in the original inoculations. The results of 
reinoculation w T ith these strains are shown in Table VII. 

From Table VII and by comparison with Table VI it appears — 

(1) That in one experiment each on jack pine and red pine the reisolated 
Rheosporangium strains gave positive results. In a second experiment on jack 
pine (No. 67) the difference between the Rheosporangium pots and the con- 
trols was not significant. 

(2) That, as in Table VI, the Pythium strains used proved on the whole 
decidedly more parasitic than the Rheosporangium strains. In experiment 66 
this is not shown by the percentage of seedlings damped-off, but is sufficiently 
evident when the germination loss as well as the subsequent dainping-off per- 
centage is considered, the survival being here, as in most other cases in which 



58 



BULLETIN 334, U. S. DEPARTMENT OF AGRICULTURE. 



either of the groups of pots compared is seriously affected by parasites, the 
most convenient index of the comparative activity of the fungi used. In such 
a comparison as thai between the Rheosporangium pots ;in<i the controls in 
experiment 68 (Tabic Yin. accidental variations in emergence, of course, over- 
shadow the slighi effect of the fungus, and the definitely determinable per- 
centage of loss alter emergence is the only value which can serve as a basis for 
any definite conclusion. 

Table VII. — Results of inoculations mi pine seedlings with initial and reisolated 
strains of Rheosporangium apJianiderniatus compared with parallel inocula- 
tions with I'lithiiuii debar yanum. 





Reisolation source. 


Num- 
ber of 
pots. 


Num- 
ber of 
strains. 


Results. 


Experimeni number, host , and 
inoculating fungus. 


Emerged 

(per 5-pot 

unit). 


Damp- 

ing-ofl 

(per 

cent). 


Survival 

(per 5-pot 

unit). 


No. 06, Finns banksiana: 
Rneosporangium — 

Strain 229 


Edson from beet 

do 


5 

5 
10 
15 
15 


1 
1 
2 
3 
3 


63 
80 
78 
63 

72 


9 
52 
43 

43 
42 


58 




39 


Strains 403 and 404 

Strains 405, 406, and 407... 
Strains 417, 430, and 433... 


SI rain 229 from beet 
SI rain 351 from beet 
SI rain 351 from pine 


44 
38 
41 




50 


10 


70 ±2. 7 


40 


42 ±3. 8 










25 

235 


47 


75 

43 


15 

33 


64 






29 




Edson from beet 

do 




No. 67, Pinus banksiana: 
Klieosporaiiuium — 

Strain 229 


5 
5 

10 
15 
15 


1 

1 
2 
3 
3 


107 
88 
91 
92 

83 



6 

s 
4 
3 


107 




83 


Strains 403 and 404 

Strains 405, 406, and 407 . . . 
Strains 417, 430, and 433... 


SI rain 229 from beet 
Strain 351 from beel 
Strain 351 from pine 


84 
88 
80 




50 


10 


90±2. 4 


4 


86±2. 9 










23 
235 


47 


87 
51 


5 
26 


83 






38 




do 




No. 68, Pinus resinosa: 
Rheosporangium— 

Strain 229 


5 
5 
10 
15 
15 


1 
1 
2 
3 
3 


105 
124 
102 
105 
100 




1 
5 
3 


105 


Strain 351 


124 


Strains 403 and Kit 

Strains 405, 406, and 4(17. . . 
Strains 417, 430, and 433... 


Strain 229 from beet 

Strain 351 from beet 

Strain 351 from pine 


101 
100 
97 




50 


10 


105 ±2. 3 


2 


102±2.5 










18 
235 


47 


104 
86 




.'7 


104 




4 


63 









A frequency graph based on the survivals of the 50 individual pots 
inoculated with Rneosporangium in experiment 68 yields a rather 
interesting asymmetrical curve (fig. IT). The shape of the curve 
is taken as indicating that in a large number of the pots the inocula- 
tion produced no effect, while in the smaller number of pots in which 
the inoculation apparently " took," the loss was rather heavy. This 
is a rather common phenomenon in inoculations which are onl} T 
partly successful, part of the pots being free or practically free from 
loss, while others are nearly cleaned out. It will be seen again in 



DAMPINc-OFF TN FOREST NURSERIES. 



59 



figure L8. This suggests, further, that pari of the lack of activity 
was due to the failure of the fungus to maintain itself vigorously 
in the soil till the pines reached a stage of sprouting in which they 
could be readily attacked. Direct inoculations after the seed starts 
to sprout arc therefore desirable to supplement the experiments 
reported. The survivals in the controls did not show any such 
asymmetrical dist ribution. 

While the Rheosporangium has given rather definite evidence of 
parasitism on Pinus banksiana under favorable conditions, the 
activity of the strains available has been much less than that of the 
Pythiwm debaryanum strains. In view of the fact that the fungus 
has not so far been isolated from pine it can be concluded to have no 
general importance 
in pine seed beds. Its 
very rapid growth on 
prune agar makes it 
very easy to isolate 
when present. 

PHYTOPHTHORA SPP. 

Phytophthora fagi 
R. Hartig has been 
commonly reported 
as the cause of death 
of seedlings of va- 
rious plants in Eu- 
rope, including con- 
ifers and herbaceous 
plants as well as 
beech (5, 8, 15, 55, 
56, 57, 59, 73, 104.) 
It has been grouped 
with the rather indefinite Phytophthora omnivora and with P. 
cactorum, the enemy of cactus, ginseng, and other plants. Wil- 
son (147, p. 54) considered it distinct, but Rosenbaum (114). 
in his biometric comparison of Phytophthora cactorum and a 
single strain of P. fagi, failed to find significant morphological 
differences. If P. fagi is even physiologically different from 
the American strains of P. cactorum, its introduction into the 
United States is to be guarded against. There is certainly no 
fungus in the United States causing the damage to coniferous 
seedlings which European reports have attributed to P. fagi there. 
As P. fagi attacks roots, it presumably can be carried in soil as well 
as on plant parts. 




If, IB 22 25 28 31 

3E£DUA/GS SURVIVING P£f? POT 

Fig. 17. — Diagram showing the results of inoculation of 
Pinus resinosa seedlings with Rheosporangium aphanidcr 
hiatus, as indicated by the number of seedlings surviving 
in inoculated pots (solid line) and control pots (broken 
line). The shape of the curve for the inoculated pots is 
taken as indicating that a large proportion of them 
were entirely unaffected by inoculation, while those which 
were at all affected suffered considerably. This is a 
frequent result in inoculations with weak parasites added 
at the time of sowing the seed. 



00 



BULLETIN 934, r. s. DEPARTMENT OF AGRICULTURE. 



A test made on jack pine with a culture of Phytophthora cactorum^ 
furnished by the department of plant pathology of Cornell Univer- 
sity, resulted negatively. At the time of sowing the seed three pots 

were inoculated with cultures on nutrient agar inserted at several 
points in each pot. After emergence additional fragments of prune- 
agar cultures were placed in contact with the seedlings, and they were 



30 


1 

- 




POTS W/T/7 

m /°yvw/(/At 


C 


,---\ i - 














20 


V 






A 












\ \ 




/ 1 




\ \ 




i 1 




% \ 




/ 




% \ 








\ \ 


to 


_ / 1 




\ \ 


D 


\ 

1 ! 1 


1 




4C 


1 
1 
1 

/ X 

/ ' s 




/=>ots w/thoc/t 

\ /=>YTHtUM 


30 


/ 1 / 


% 






' / 


X 






> / 




\. \ 




/ / 








/ /■ 








/! 




\ \ 
\ \ 




' / 




\ \ 


SO 


/ / • 




\ \ 




/ / 




\ \ 




/ / 




\ \ 








\ \ 




* / 1 
/ / 1 




\ \ 
\ \ 


JO 


/ * 


fc 


X \ 






1 


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/ I 


^ s **~ 


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/ 1 1 ! 




1 1 ^***^vj 



0-/4- 



/S-24- 30-4-4- 4-SS9 60-74- 76-89 
<S£-£T£>L//V(?S S(/#{//y//VG P£/?POT 



90-/04- 



Pig. 18.— Frequency of pots with different numbers of surviving seedlings of Pinus 
banksiana, Inoculation experiment No. 31. The solid lines represent pots to which 
cultures of saprophytic organisms were added. The broken lines are based on pots to 
which no saprophytes bad boon added. The solid lines are based on 78 pots in the 
upper graph and 80 pots in the lower one; the broken lines on 33 pots in the upper 
and 25 pots in the lower graph, Pythium debaryanum was added jus) after sowing the 
seed at a single point in each pot represented by the two upper linos. Cultures of 
saprophytes were applied broadcast two days before the Pythium inoculations were made. 

sprayed with a spore suspension. The pots were covered with glass 
to increase atmospheric moisture, and the seedlings were occasionally 
sprayed with an atomizer. The soil was an autoclaved mixture in 
which simultaneous inoculations in a different room with Pythium 
and Corticium proved successful. The failure of the Phytophthora 
may possibly have been due to the lower temperature at which the 
pots inoculated with it were kept (15° to 20° C.). 



DAMPING-OFF IN FOREST NURSERIES. 61 

A species of Phytophthora was isolated by Mr. R. (1. Pierce from 
damped-off Pinus resinosa in Minnesota ami used in four inocula- 
tion experiments, the results of which appear in Table VIII. In the 
first of these experiments unboiled water was used on the pots, and 
mice obtained access to the pots of the second test. Probably as a 
result of these things infection occurred in the controls in both cases, 
and the results were inconclusive; in the later experiments these 
two sources of infection were eliminated, and in experiments 68 and 
72B the controls were free from disease. Parasitic activity was in- 
dicated rather strongly in experiments 68 and 72 (on P. resinosa and 
P. banksiana) and to a certain extent in experiment 66. In experi- 
ment (>7 it was evident that the Phytophthora was nearly or entirely 
inactive. Comparison of the results in experiments 66 and 68 with 
the results from inoculations with Rheosporangium aphanidermatus 
in the same experiments (Table VII) suggests that the Phytoph- 
thora may be better able to attack the pine from which it was isolated 
than the Rheosporangium, while the latter fungus caused consider- 
ably more destruction to Pinus banksiama than the Phytophthora. 
Comparison of the results in the pots inoculated with Phytophthora 
and those inoculated with Pythium debaryanum in all the experi- 
ments indicates that the Phytophthora strains used were less virulent 
than most of the strains of P. debaryanum and very certainly less 
destructive than the most dctive strains of either P. debaryanum or 
Corticium vagum. This species of Phytophthora has been reisolated 
from damped-off Pinus ponderosa in experiment 72. 

Direct inoculations of the stems of seedlings of Pinus resinosa soon 
after they emerge from the soil have so far confirmed the lack of 
parasitism of Phytophthora cactorum and of the cultures of Phy- 
tophthora sp. grown by the writer. The identity of this species has 
not yet been determined. It is able to grow only about one-fourth as 
rapidly as Pythium debaryanum on the medium which has been 
used for isolation and may therefore be more common in the seed 
beds than the small number of isolations by the planted-plate method 
would indicate. However, its oospores, larger and darker than those 
of Pythium debaryanum (usually over 20 [x in diameter), should have 
been recognized in the routine microscopic examination of planted- 
plate cultures had this species been frequently present, even if it 
had not grown fast enough to get out ahead of the other organism 
and allow isolation. It is not believed that it is common enough in 
pine seed beds to be of importance, even if other strains should be 
found more virulent than those which have been available. 

MISCELLANEOUS PHYCOMYCETES. 

A fungus, apparently referable to the somewhat indefinite Pythium 
artotrogus (Mont.) De Bary, w r as isolated by Mr. Glenn (x. Hahn 
from Pinus resinosa in Michigan and from damped-off Pinus bank- 



62 BULLETIN 934, 0*. S. DEPARTMENT OF AGRICULTURE. 

siana in pots of autoclaved soil which had received tap water at 
Washington, D. C. It agreed both in the appearance and measure- 
ments of its spiny oogones and smooth oospores with Pythw/m arto- 
trogus (P. hydnosporus) as described and figured by Butler (23). 
In addition to the spores which Butler describes, there appeared in 
apparently pure prune-agar cultures of different strains bodies with 
smooth walls, of somewhat irregular ovoid outline, and mostly larger 
than either oospores or oogones. They are very much less abundant 
than the sexual spore forms. Their greatest diameter varied from 
11 \x. to over 40 \).. The germination of these bodies was not observed. 
Efforts to induce the fungus to produce swarm spores by growing 
them in liquid nutrient media and transferring them to pure water 
were unsuccessful. This failure to produce /oospores is further in- 
dication of the identity of the fungus with that described by Butler, 
who says that asexual reproduction is unknown. 

The strain from Michigan was a rather weak growing organism, 
difficult to maintain in tube cultures without rather frequent trans- 
fers. Its parasitic activity in the experiments reported in Table VIII 
is nil or negligible. Because of the poor seed and small number of 
seedlings involved in experiment 72B, the percentage of damping- 
off there given means only a single seedling dead. The Washington 
strains, on the other hand, though evidently not strong parasites, did 
apparently cause the death of a number of seedlings. The best evi- 
dence of this is in experiment OS. in which there was damping-off 
in each of the live 5-pot units containing the Washington strains and 
none in any of the IS control pots. The available strains were less 
active not only than PytMum debarymyu/m^ but less than the Rheo- 
sporangium and Phytophthora strains used. The fungus is be- 
lieved to be a potential parasite on [tine seedlings, but not one of any 
general importance. What is probably the same fungus had ap- 
peared in the writer's cultures from western nurseries in conjunc- 
tion with /'. (lelxtri/aitum, but not commonly, and it had not been 
isolated. While its growth rate is only about half that of P„ debartj- 
anum on prune agar, it is nevertheless so much faster than that of 
many fungi that it should have been more often obtained in culture 
were it at all common in damped-off seedlings. 

Another fungus, presumably an oomycete but producing only 
chlamydospores in the writer's cultures, was obtained from damped- 
off olive seedlings furnished by Prof. W. T. Home and from soil 
direct, both at Berkeley, Calif. The fungus is apparently the same 
as one which has been occasionally seen in cultures from pine seed- 
lings in the Middle West, but had not before been isolated. The 
hyphse are ordinarily nonseptate, and the growth on corn-meal agar 
is superficially much like that of Pythium debaryanum, but with 
greater tendency toward local zonation and aerial growth and less 



DAMPING-OFF IN FOREST NURSERIES. 



63 



than half as rapid. Chlamydospores are mostly intercalary, at first 
subspherical, soon becoming polygonal, and after a Eew days they 
shrivel and exhibit thick, angular walls. In size the unshrunken 
spores usually lie between 8 and L2 y. in diameter, but bodies as 
large as 20 \x occasionally occur. Antheridia have not been observed, 
and the shriveled bodies are not believed to be oospores, though the 
observations made have not been sufficient to exclude such a possi- 
bility. No other spore form was obtained in water culture, using 
various nutrient substrata. In inoculation the strain from olive (the 
"undetermined Phycomycete " included in Table VIII) has given 
negative or nearly negative results in three inoculation tests in which 
other fungi gave positive results. In a test not included in the table, 
in which Firms ponderosa was the trial host, damping-off was slightly 
higher in the inoculated pots than in the controls, but the difference 
was apparently due to accidental infection with Botrytis and 
Pythium debaryanum. As all the seedlings in pots inoculated with 
P. debaryam/um in this additional experiment were killed, the rela- 
tive unimportance of this strain of the small-spored fungus was 
further indicated. An additional test of both the olive strain and 
the strain from soil was made by inoculating seedlings of Pinus 
han/i'siana and P. ponderosa growing on filter paper in Petri dishes. 
Some of these were kept wet with water, some with an inorganic 
culture solution, and some with the inorganic solution plus peptone 
and dextrose. Agar cultures were applied directly to the seedlings. 
The seedlings inoculated with the small-spored fungus remained 
alive as long as the control seedlings, while parallel inoculations with 
Pythium debaryanum resulted in the early decay of the seedlings. 

Table VIII. — Results of inoculations with miscellaneous oomycetes on pines in 

autocluved soil at the time of soicing. 

[In all the experiments included in this table, the inoculum consisted of fragments of agar cultures 
distributed with the seed at one side of each pot over about one-fourth of the pot area. The controls 
received sterile agar in the same way.J 





Num- 
ber of 
pots. 


Results. 


Experiment number, host, and inoculating fungus. 


Emerged. 


Bamp- 

ing-on". 


Survival. 


No. 66, Pinus banksiana: 
Phytophthora sp.— 


5 

5 
5 
4 
5 
25 

5 

5 

5 

S 

23 


Per B-pot 

ii nil. 
70 
75 
94 
115 
83 
75 

96 
88 
99 
102 
98 
87 


Percent. 
30 
28 
16 
14 
9 
14 

1 
3 

2 

5 


Per S-pot 

unit. 

40 


Strain 372 


54 




79 




99 




76 




64 


No. 67, Pinus banksiana: 

Phytophthora sp. 


95 




85 


Strain 375 


99 




Hid 




98 




83 



64 



Bl LLETI2S 934, U. S. DEPARTMENT OF AGRICULTURE. 



Table VIII. 



Results of inoculations with miscellaneous oomycetes on pines iii 
autoclaved soil at the time of solving Continued. 





Num- 
ber of 

pots. 


Results. 


Exp rimenl number, host, and inoculating fungus. 


Emerged. 


l>ani|>- 

ing-ofT. 


Sun i\ai 


No. fis, Finns resinosa: 
Phytophthora sp. — 

Strain 35s 


.") 
5 
5 
5 

5 
5 
5 
5 
5 
5 
IS 

3 
3 

2 
3 
3 
16 

3 
3 

3 
2 
2 
14 


/■. i .' pot 

unit. 
10! 
109 
98 
121 

122 

120 
96 

110 
94 
84 

104 

Per S-pot 

n nil. 
20 
62 

45 
37 
40 
35 

8 
13 

11 

29 

6 

9 


r, rc< in. 
7 
18 


Per n-ix>t 
n nil. 
97 




RQ 






Pythium artotrogus (?), Michigan strain 




1 
9 
5 
6 
1 
2 


35 


7 

25 
5 

50 
8 


6 




121 


Pythium artotrogus (?), Washington, l>. e. 

St rain vji 


121 


Strain 82S 


109 


Strain 83] 


in 


Strain 832 


103 


Strain RH3 


93 


Undetermined Phycomycete 


82 


Controls 


mi 


No. 72 A. Pinus resinosa: 
Phytophthora sp. — 

'Strain:;:.'. 


Per .1-pot 

unit. 
13 


Pythium artotrogus (?), Michigan strain 

Pythium artotrogus (?), Washington, !>.('. 
Strain 821 


62 
42 


St rain 831 


37 


Strain 833 


30 


Controls 


33 


No. 72B, Pinus pondcrosa: 


4 




12 


Pythium artotrogus ('.'). Washington, D.C. 

Strain 821 


11 


Strain 833 


27 
6 




9 







OTHER FUNGI. 

Data on the possible relation between various other fungi and the 
damping-off of conifers have been already summarized by Hartley, 
Merrill, and Rhoads (68, p. 546-550). Pestalozzla funerea on the 
basis of the experiments of Spaulding (135), Botrytis cinerea on the 
basis of observation and very preliminary inoculations, and Tricho- 
derma koningi on cultural evidence only are all believed to be po- 
tential causes of damping-off, though not ordinarily important. Al- 
ternaria sp. is under a certain amount of suspicion on account of its 
frequent association with the damping-off of conifers, but it has 
never been used in experiments. Rhisopus nigricans (incorrectly re- 
ported as Mucor), Trichothecium roseum, RoseJlinia sp. from nursery 
soil, Chaetamium sp. from maple roots, strains of Penicillium and 
Aspergillus, Phoma betae, and Phoma spp. are all reported to have 
been used in inoculations with negative results. 

Since the publication of the above summary a preliminary success- 
ful inoculation experiment with Botrytis dnereaon recently emerged 
Pseudotsuga taxifolia has been found briefly mentioned in an article 
by Tubeuf (140) on another disease. Further experiments with va- 



DAMPING-OFF IN FOREST NURSERIES. 65 

rious strains of Botrytis, both from conifers and from other hosts 
(the latter supplied by the departments of plant pathology of the 
California and New York (Cornell) Agricultural Experiment Sta- 
tions), have already yielded confirmatory evidence of the parasitism 
of B. cine red. 

"While a considerable number of fungi have been considered in the 
foregoing, it is entirely possible that there are still parasites which 
have received no consideration and that some of them may perhaps 
be important. The moist-chamber method of culturing parasites 
for isolation yields only those which produce spores readily; the 
planted-plate method is not well adapted to the isolation of slow- 
growing fungi or bacteria. It is suggested that in further culture 
work with damped-off conifers an attempt be made to secure slow- 
growing organisms by dilution plates of teased-up fragments of 
recent lesions. 

RELATIVE IMPORTANCE OF THE DAMPING-OFF FUNGI ON 

CONIFERS. 

The relative importance of the different damping-off parasites is 
something that has not been thoroughly investigated for any host. 
The most information on this point is that given by Busse, Peters, 
and Dlrich (22) for sugar beet. In this case they find the special- 
ized Phoma beto-e distinctly the most important, with Pythium de- 
baryanum second and Aphanomyces levin third. 

Peters (l ( "i) apparently considered Rhizoctonia unimportant as 
a cause of beet damping-off. The opposite was indicated by a small 
number of cultures by Edson (38) from beet seedlings on Kansas 
and Colorado soil. These yielded more Corticium vagum than any 
other parasite- and no Pythium at all. Johnson (81) states that 
most of the damping-off of tobacco seedlings is due to Pythium 
debaryanum and Corticium vagum. Atkinson (1), speaking for 
cotton in Alabama, and Sherbakoff (127, p. xcv; 128; 129), speak- 
ing for truck crops in Florida, make Corticium vagum the impor- 
tant damping-off parasite, with P. debaryanum negligible. Home 
(oral communication) found the same situation in tobacco seed 
beds in Cuba. Atkinson (3), in an article on trees, held that many 
of the cases of damping-off attributed to P. debaryanum are in real- 
ity due ta C. vagum. Peltier (98, pp. 336-337) has reported Rhi- 
zoctonia solani as the cause of damping-off of a large number of 
plants, recording his observation of the damping-off of seedlings of 
nearly 50 species of miscellaneous genera and cuttings of 13 different 
species, all of which he attributes to the Rhizoctonia. He does not 
state whether in this case he used diagnostic methods likely to de- 
tect Pythium debaryanum if it had been present. 

19651°— Bull. 934—21 5 



66 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

For the conifers, no very reliable data on relative importance have 
been published. Numerous European reports emphasize the damage 
due to Fusarium spp., while a smaller number attribute loss to 
Phytophthora fagi or to both. There seems to have been little effort 
to determine the presence or absence of Corticium or Pythium, so 
these reports can not be given great weight. Spaulding's evident 
belief (136) in the importance of Fusarium has more weight, as ho 
was on the lookout for the other fungi ; the moist-chamber diagnostic 
method employed in most of this work was, however, not Avell 
adapted to the detection of either one. The same is true of the work 
of Eathbun (106), in which dilution plates of seed-bed soil were 
employed. Rankin (105) attributes to Fusarium spp. the greatest 
importance in tree seed beds in this country, with Pythium debary- 
anum and Rhizoctonia spp. important in certain cases. Gifford (46) 
emphasizes the importance of Fusarium, while Clinton (28) appar- 
ently found Khizoctonia {Corticium vagum) especially prevalent 
in the examinations he made. 

On the basis of the data presented or summarized in this bulletin, 
it is believed that of the various organisms which have been con- 
nected with damping-off in coniferous seed beds Pythium, debary- 
anum, Corticium vagum, and Fusarium spp. include all of impor- 
tance. The others, either because of low indicated virulence or 
infrequent occurrence, and in most cases both, do not seem to merit 
extensive consideration. 

In order to form an idea of the relative frequency of the parasites 
named above as important, there have been brought together in 
Table IX the results of the examination of 438 damping-off foci in 
untreated beds and 304 foci which have appeared in beds which had 
received various disinfectant treatments. The data are presented 
by foci rather than by individual seedlings, as was done in the census 
reported by Busse and his coworkers. Most of the diagnoses were 
made by planting recently diseased seedlings in plates of solidified 
prune agar, all the seedlings taken from the same focus, or " patch," 
of damped-off seedlings being put into the same Petri dish. The 
resulting growth was in some cases transferred to a tube for later 
examination, but was usually examined directly in the plate. In a 
smaller number of foci the seedlings were macerated and examined 
directly without recourse to culture methods. As Pythium debary- 
< mum does not commonly fruit in diseased seedlings of pine or of 
tobacco (81) and its hyphse are both difficult to find and not in 
themselves considered a sufficient diagnostic character, this latter 
method of examination is not so satisfactory for the determination of 
Pythium as it is for Corticium, which is easily recognized by its 



DAMPlNC-oi-T IN FOREST NURSERIES. 



67 



thick-walled truncate-tipped hyphae and characteristic branching. A 
further difficulty in the direct-examination method, unless the seed- 
lings are sectioned, is in distinguishing between Corticium hyphse 

which are in the tissues and those outside. The well-known habit 
of the Corticium of sending hyphae superficially over the surface of 
plants which it is not appreciably injuring makes it evident that only 
hyphae actually found in the tissues have diagnostic value. Direct 
microscopic examination is. furthermore, very likely to fail to detect 
Fusarium. The planted-plate method therefore appears the better of 
the two. and the results of the culture diagnoses appearing in the 
lowest two lines of Table IX deserve probably more attention than 
the total occurring a few lines above, in which the results of direct 
examination of the seedlings are also included. The high proportion 
of Corticium reported from the Michigan and Minnesota nurseries 
is probably due in part to the fact that most of the examinations 
made there were of the direct microscopic type. 



Table IX. — Results of the examination of damping-off foci in coniferous seed 
beds for Pythium dciaryanum, Corticium vagum, and Fusarium xin>. 





Untreated beds. 


Beds of heated 
soil. 


Beds treated with 
strong acids. 


Grouping. 


•6 

c 

a 

03 

a 

Cj 

o 
ft 


Number 
showing — 


•6 
s 

. el 

a 

03 
X 

'o 

O 
ft 


Number 
showing — 


•6 

CO 

.9 

9 

'3 
o 

ft 


Number 
showing — 


• 




3 

3 
>> 
ft 


a 

3 
'P 

u 
o 
O 




3 
03 
3 
ft 


a 

3 

3 
ft 


a 

3 

|3 

u 

o 
O 


OS 

B 
ft 


i 

3 

3 

>. 

ft 


a 

3 

]3 

o 
O 


a 

3 

*n 

03 

3 
ft 


By locality: 
Berkeley, Calif 


4 

22 
34 

18 

20 

224 

42 

4 

45 
13 
12 


3 

6 

4 
4 
124 
15 


14 
11 
3 


1 

19 
20 



9 

45 

21 

4 

33 

7 
3 


2 

13 

9 

7 

155 

5 

1 

2 
1 

9 










5 


5 





2 
































Garden City, Kaiis. — 


14 
15 
28 


5 
2 

10 







7 
6 
17 


28 

16 

99 

1 


8 

9 

34 




2 
2 
2 



q 




8 




61 




n 


Dundee, 111 












East Ta'was, Mich. — 










1 
13 


1 

7 



6 


o 












? 




7 


2 

19 
30 








3 

33 
52 








64 
39 

6 

38 

58 
39 


12 
5 

8 
50 

4 
3 




Total: 


438 
100 


184 
42 


1(12 
37 


204 
47 


64 
100 


163 
100 


82 




50 






By diagnostic methods: 
Direct examination — 


156 
100 

2S2 
100 


39 

25 

145, 
51 


108 
69 

54 
19 


25 

16 

179 
63 





16 
100 

147 
100' 


4 










?•> 


l 'lanted-plate cultures- 


64 
100 


19 
30 






33 
52 


7S 




53 



68 



IHLLETIN 034, U. S. DEPARTMENT OF AlilU CULTURE. 



Table IX.- — Results of the examination of damping-off foci in coniferous seed 
beds for Pythium debaryanum, Cortieium vagum, and Fusarium spi>. — Con. 





Beds treated with 
formaldehyde. 


Hods treated with 
copper sulphate. 


[Beds i reated with 
zinc chlorid. 


All treated beds. 


Grouping. 


■6 
e 

a 
1 

y, 

Cj 

'o 
o 


Number 

showing — 


•6 

a/ 
| 

1 

03 

y. 
oj 

*o 
o 

Pi 


Number 
showing — 


■d 

o> 

3 • 
3 

03 

y. 

'3 

o 

Pi 


Number 
showing — 


■6 

9 

.3 

S 
a 

o 

'o 

o 

Pi 


Number 
showing — 




3 
Pi 


u 

o 
o 


03 

3 


3 
B 

PL) 


u 

o 


g 

.3 

'C 
03 

9 

Pi 


s 

Pn 


i 

u 
o 


3 

Pi 


a 

3 

2 
5 


£ 

3 
|o 

Ei 
o 
O 




a 

3 

03 

3 

Pi 


By locality: 
Berkeley, Calif 






5 


? 
































































Garden Oily, Kans. — 
Garden City Nurseries . . 
Kansas Nurseries (sand) 


6 





6 


3 


1 








1 


3 


1 





3 


52 
31 
175 

1 


14 

11 

67 



8 
2 
3 



23 
14 


31 


20 


1 


24. 





3 





5 


8 








5 


112 



































East Ta'was, Mich.— 


























1 

32 
7 


1 
20 
2 



13 






East Tawas Nurseries. . . 


8 


5 


4 





6 


5 


3 


3 


5 


3 





2 


7 
3 






























Total: 

Nnmher ■ 


48 
100 


25 

52 

5 
(13 

20 
50 


11 
23 

4 
50 

7 
18 


27 
.56 




27 
08 


13 
100 


8 
62 

4 
80 

4 
50 


3 
23 

2 

40 

1 
13 


9 
69 

2 
40 

7 
88 


16 
100 


4 
25 

3 

CO 

1 

9 












10 
63 

2 

40 

8 
73 


304 
100 


120 
39 


20 

9 

14 
41 

12 
4 


101 




53 






By diagnostic methods: 
Direct examination — 


8 
100 

40 
100 


5 
100 

8 
1(10 


5 
100 

11 
100 


34 
100 

270 

100 


18 
53 

102 

38 


8 




24 


Planted-plate cultures — 


153 




57 







The data on the different nurseries do not allow any generalizing 
on the basis of locality except to say that all of the fungi seem quite 
generally distributed in the Lake States and Great Plains region. 
In general, it appears that the Fusaria as a group are more common 
than either of the other fungi ; as they grow more slowly than either 
the Pythium or the Cortieium, they were probably rather more 
common relatively than even the plate-culture method indicated. 
It also appears that the Pythium occurred in more foci than the 
Cortieium in the beds examined. Further culture work, perhaps 
by the method of dilution plates of fragments of lesions, seems de- 
sirable, especially in the East and the Northwest, regions in which 
there are large coniferous nurseries and in which nothing like a 
parasite census has been attempted. Observations on the type of 
focus occurring in most of the nurseries in the Rocky Mountains 
leads the writer to believe that Cortieium will be found especially 
important there. 

While the data on the fungi in foci in disinfected beds are insuffi- 
cient to serve as a basis for much in the way of conclusions for any 
individual treatment, they in general agree with the assumption, 
which knowledge of the fungi would favor, that Cortieium is the 



DAMPING-OFF IN FOREST NURSERIES. 69 

most easily controlled by soil disinfection (see the bottom line in 
the last four columns of Table IX). Its poor adaptation for aerial 
dissemination would lead one to expect to find it seldom in beds 
treated with efficient disinfectants. The entire absence of Corticium 
in heated soil therefore seems somewhat significant. The rather 
high Corticium yield in the formaldehyde plats is of some interest 
in view of the reported inefficiency of formaldehyde in destroying 
Corticium vagumon. potato tubers (48, 50). As will be noted from 
the data given, more than one suspected parasite was often found in 
what appeared to be a single focus. This was probably in some 
cases due to independent foci being nearly concentric ; it also in 
some cases undoubtedly means that one of the organisms found was 
only secondary. In the beet-seedling cultures by Pmsse and bis 
associates, individual seedlings yielded two or more potential para- 
sites in 100 of their nearly 1,300 examinations. It not infrequently 
happened in the work on pine seedlings that no fungus recognized 
as a likely parasite could be isolated. This was especially true 
in plate cultures when Rhizopus or Trichoderma happened to be 
abundant, as both are very fast growing and often suppress para- 
sites. This is an additional reason for the development of some 
method as a dilution plate of lesion fragments for diagnosing damp- 
ing-off. 

Even an accurate and complete census of the organisms present in 
the different foci could not be directly interpreted in terms of rela- 
tive importance. None of the parasites so far used in inoculation 
have been vigorously parasitic under all conditions. Of both Corti- 
cium vagum and Pythium dclmryanum some strains, microscopically 
indistinguishable from the others, are very weak as parasites. Only 
part of the Fusarium species are parasitic on pine, and data showing 
which are and which are not parasitic are known for only a very 
few. There is therefore no fungus which can be said positively to 
be the cause of any particular damping-off " patch " simply because 
it was found in some of the dead seedlings in the patch. In an occa- 
sional exceptional case, such as the large Corticium patch in figures 
7 and 8, there is such a vigorous growth of the fungus that its pre- 
dominance is undoubted, but such cases are rather rare. A census 
throws light on the importance of the different fungi, but can be 
interpreted only in the light of inoculation results. 

For Pythium and Corticium the inoculation data do not permit 
any simple comparison between the two, for the reason that neither 
is uniform. Each has strains of high virulence and strains having 
practically no effect on pines. In the inoculations in autoclaved soil at 
sowing time the strongest strains of Corticium vagum have on the 
whole caused more damage than any of the Pj'thium strains, but, on 
the other hand, there has seemed to be a higher proportion of very 



70 r.ru.KTix 034, U. s. DEPARTMENT ok AGRICULTURE. 

weak strains of C. vagum than in the case of Pythium. Tn inocula- 
tions on Pinus banksiana and P. ponderosa in Kansas sand treated 
with acid followed by lime, the average Corticium was very much 
more destructive than even the strongest Pythium strains, allowing 
practically no germination in most cases. On the other hand, in ex- 
periments in which the inoculum was applied directly to Phn/s 
resinosa and P. ponderosa seedlings, either immediately after germ- 
ination or after the older parts had become resistant, the Pythium 
has been the more effective. The inoculation evidence so far avail- 
able justifies so nearly equal emphasis on the two that it can prac- 
tically l>e eliminated from the calculations. It is the waiter's opinion 
that the Corticium strains are probably rather less virulent on the 
average than the Pythium strains, but perhaps better able to main- 
tain themselves and spread from one seedling to another in most 
soils. The evidence of Table IX that the Corticium seemed less fre- 
quent in the damping-off foci is more or less counterbalanced by the 
apparent larger size of many of the disease patches which it seems 
to cause in the seed beds. Nearly all the large clean areas such as 
are shown in figures 7 and 8 have been found to contain abundant 
Corticium hyphae. The evidence on the whole seems to indicate a 
very nearly equal importance for the two fungi. The Pythium is 
probably somewhat the more important for the stations at which 
most of the cultures in Table IX were made, but the Corticium has 
received more emphasis from other observers in this country and is 
indicated by the writer's observations to be more important in the 
western mountains than any other damping-off fungus. 

The inoculation evidence for Fusarium spp., though less complete 
than for Corticium and Pythium, is nevertheless rather helpful in 
indicating their importance rating. Xone of those so far tested in 
inoculations at sowing have shown the destructiveness of the aver- 
age strains of Pythium or of the stronger strains of Corticium ; while 
this is only in part a test of virulence and in part a test of the 
ability of the fungus to grow saprophytically in the soils used, the 
indication is that no one Fusarium species is the equal in destructive 
capacity of either Corticium vagum or Pythium debaryanum. How- 
ever, when all of the Fusarium species which occur in the seed beds 
are considered, the group as a whole may prove quite as important 
or even more important than either of the other two fungi. The data 
already at hand rather definitely indicate considerable importance for 
all three. 

DAMPING-OFF FUNGI AS CAUSES OF ROOT-ROT AND LATE 

DAMPING-OFF. 

As has been already stated, root-rot, often with frequent recovery, 
has been commonly observed in seedlings several weeks old. It has 
been especially common in the vicinity of old damping-off foci in 



DAMPING-OFF IN FOREST NURSERIES. 71 

which Corticium vagv/m appeared to be the active parasite, but 
beyond this indication of the causal relation of C. vagurn it was not 
known which of the damping-off fungi were able to attack the roots 
of seedlings too old to be killed by damping-off. To throw light on 
this point, seedlings of Pinus ponderosa and P. resinosa grown in 
autoclaved soil in the greenhouse and approximately H months old 
were inoculated with different fungi. There had been a certain 
degree of early damping-off in these pots, but it had apparently 
ceased before the inoculations were made. The inoculum used con- 
sisted of cultures on rice introduced through the drainage holes at 
the bottoms of the pots. The strains of Pythium debaryanum and 
Corticium vagum used were the ones which had given maximum 
results in earlier inoculation experiments at the time of sowing. The 
strain of Fusarium ventricosum was the only one available, and the 
Fusarium monilifornu strains were all of approximately equal viru- 
lence, the three used having given as much evidence of parasitism 
as any of the strains of this species in the earlier damping-off experi- 
ments. Three pots of each pine were inoculated with each strain. 
Two 3-pot units of each pine were set aside as controls and inoculated 
with sterile rice. In addition, three pots of each pine were kept in 
the same bench without the addition of any inoculum, for comparison 
with the controls with rice. The results of this experiment, taken 
a month after the inoculations were made, with the seedlings averag- 
ing 2% months old, appear in Table X. The roots of the living seed- 
lings were washed out carefully with water to permit examination. 

The results in so far as they indicate root-rot of the oldest seedlings 
are. best shown by the figures in columns 4 and 5. These seedlings 
were so far advanced that the fungi had not been able to kill them, 
and nearly all would probably have recovered if they had not been 
dug up. It will be noted from column 4 of Table X that a consider- 
able portion of the Pinus ponderosa seedlings with root-rot had al- 
ready made their recovery apparent by pushing out adventitious 
roots above the decayed portion at the time they were examined. 

For Fusarium ventricosum, there was only the merest indication of 
ability to attack pine roots at this stage. For F. moniliforme the 
evidence is somewhat better, more pots being included and the dif- 
ference in healthy-topped seedlings with injured roots between the 
inoculated pots and the controls being approximately twice its indi- 
cated probable error for each species. The percentage of root-injured 
seedlings in the Pythium debaryanum pots exceeded that in the con- 
trols in each species by between three and four times the probable 
error of the difference, while the difference in percentage between 
the Corticium vagum pots and the controls is approximately four 
times its probable error in the case of Pinus ponderosa and five and 
one-half times its probable error in the Pinus resinosa pots. The 



72 



BULLETIN 934, U. S. DEPARTMENT OF AGRICULTUEE. 



weak point in the results is, of course, the insufficiency of the 6-pot 
and 9-pot groups as bases for probable-error determination. The 
indicated relative ability of these different fungi to cause root-rot 
is about the same as their relative ability to cause the damping-off of 
sprouting seed and young seedlings, as indicated by the results of the 
earlier experiments in which inoculations were made at the time of 
sowing. The fact that only the very strongest available strains were 
used and that the pots were rather heavily inoculated is to be kept 
in mind in considering these results. As in the seedlings examined 
in the nursery beds, when a root system was partly rotted it was 
only the younger portions of the roots that were affected. The evi- 
dence obtained from this experiment needs to be amplified by experi- 
ments with other coniferous hosts, other strains of the fungi, and 
under other conditions. The experiment just described furnishes 
the only evidence available on the relation of the important fungi 
Pytldum debaryanum and Corticium vagum, to the root-rot of conifers 
and is therefore presented as a preliminary contribution. 



Table X. 



-Results of root inoculations of older pine seedlings with damping-off 
fundi. 





Number of — 


Seedlings which developed root-rot 
(percent). 


Host and inoculating fungus. 


Pots. 


Seed- 
lings. 


Tops still healthy. 


Killed. 




Root recovery. 


Average 

of in- 
dividual 
pots. 


Total. 




Started. 


Not 
started. 




1 


2 


3 


4 


5 


(5 


7 


8 


Finns ponderosa: 

Pythium debaryanum, strains 295, 550, 

and 810.a 
Corticium vagum, strains 147, 213, and 

747. a 
Fusarium moniliforme, strains 249, 251, 

and 260.6 


9 
9 
9 

3 

6 
3 

9 

9 
8 
3 
fi 
3 


71 
56 

64 

18 
41 
18 

140 
146 
128 

39 
115 

51 


27 

16 

19 

17 
2 


3 
3 






25 

34 

27 

39 
15 
17 

16 

16 
11 
5 
3 



53±4.5 

51±3.5 

42±6.2 

50 

22±6.5 

23 

18±4.0 
21±2.4 
12±3.7 

4 

4±2.0 




4 

4 







.12 
13 
2 

6 
2 


56 
54 

45 
56 




17 




17 


Pinusresinosa: 


31 




33 




13 




5 




10 




2 







a For relative virulence of these strains on younger seedlings as compared with other strains of the same 
species, note their position in figures 11 and 14. 

t> For performance of these strains in inoculations at time of sowing, see an earlier publication (68, 
table 2). 

The figures in column 7 give information as to the percentage of 
late damping-off resulting from the inoculations. A certain per- 
centage of the early t}^pe of damping-off appeared in some of the 



DAMPING-OFF IX FOREST NURSERIES. 73 

pots, as there were still present a number of soft-stemmed seedlings 
from seeds which were slow in germinating. These younger seed- 
lings were excluded in counting the dead, the rule being to include 
only plants which had developed a sufficiently rigid stem to remain 
upright alter death. Comparison of the percentage of killed with 
the total percentage attacked for the two pines is rather interesting. 
As has already been pointed out, while Pinus resinosa suffers very 
heavy damping-off losses at a number of nurseries it seems to be 
less susceptible than some other species to parasitic injury during 
the sprouting period, before the seedlings appear above the soil sur- 
face. Observation of beds of this species during different seasons 
has indicated that it has not a greater susceptibility, but rather the 
fact that its susceptibility lasts longer, which causes it to suffer as 
seriously as it does at certain nurseries. It is indicated in Table X 
that the succulent root tips of Pinus pondcrosa are just as easily 
attacked by damping-off parasites as those of P. resinosa — in fact, 
considerably more easily attacked, as indicated by the figures in col- 
umn 8. With the P. ponderosa seedlings, however, the older parts 
of the roots had become resistant at this age in nearly all cases, while 
of the affected P. resinosa seedlings more than one-third were still 
unable to limit the lesions, and death resulted. 

In general, this experiment indicates that Corticium vagum and 
Pythium debaryamim are able to cause the death of some pine seed- 
lings which have developed rigid stems and that both are also able, 
as has been found by other workers in the case of sugar beets, to 
cause " root sickness," the rot of the younger portions of the root 
systems, in seedlings which have developed too much resistance to 
be killed. The evidence for the parasitism of the two Fusarium 
species on these older root systems is not so good; as in the experi- 
ments on younger seedlings, their ability to attack the pines is prob- 
ably less than that of the other two fungi. Further inoculation ex- 
periments are desirable both with these fungi and with others on the 
roots of seedlings too old to succumb to the more ordinary types of 
damping-off. 

RELATION OF ENVIRONMENTAL FACTORS TO DAMPING-OFF. 

In the earlier section dealing with disease control, mention was 
made of the general belief on the part of men who have had experi- 
ence with seedling diseases that damping-off is favored by thick seed- 
ing, by much organic matter, especially by poorly rotted manure in 
the soil, and by excessive moisture in the air and soil. It is also 
commonly stated that high temperature favors the disease ; on this 
point there is perhaps a less general agreement. Practically all the 
evidence on these points is observational. 



74 



BULLETIN 934, U. S. DEPARTMENT OE AGRICULTURE. 



DENSITY OF SOWING. 

The relation between the disease and thick sowing was strikingly 
indicated for tobacco seedlings in a single experiment by Johnson 
(82). For pines the only available information is from four experi- 
ments on Pinus banksiana. The results of the first two appear in 
figure 19. In both experiments there is an indication of an increase 
in the percentage of diseased plants as the seed density is increased. 
There is, however, no such marked relation as in Johnson's work. 
As the pines were sown in drills, they were so close together even in 
the less dense plats that no very great increase in the ease of spread 
i ___^__^_ - ______________^^^^____ of the disease was to 

be expected from in- 
creasing the density. 
Greater differences 
should be expected 
in broadcast beds. 
That heavier losses 
have been found in 
drill-sown beds than 
in those sown broad- 
cast (69, 139) is pre- 
sumably explained 
by the fact that with 
equ al numbers of 
seed per square foot 
of seed bed the seed- 
lings are much closer 
together in drills 
than in broadcast beds, and thus the spread of the mycelium of para- 
sites from one seedling to another is facilitated. 

Two tests of different seed densities were also made in 3-inch pots 
of autoclaved soil in the greenhouse. Each regular pot was sown 
with 28 seeds (equivalent to 000 per square foot). The pots were 
inoculated by adding to each a single small fragment of an agar 
culture of PytMum debaryanum. Uninoculated pots showed an emer- 
gence of approximately 50 per cent of the seed and were entirely free 
from subsequent damping-off in both experiments. The results ap- 
pear in Table XI. 

In this case not only the damping-off after emergence but the loss 
before the seedlings appeared bore an apparent relation to sowing 
density. In the field experiments there was no evidence that the loss 
before the seedlings appeared was affected by seed density. 




600 J.200 4300 Z/f-OO 3,000 

A/OMBSff orseeos sotv/v /=£■/? square - /vot or bed 

Fig. 19. — Diagram showing the extent of damping-off in 
drill-sown Pinus banksiana in plats with different seed 

densities. The regular seed density at tins nursery was 
dint seeds per square foot. 



DAMTMXc OFF IX FOREST NTJRSERIES. 



75 



Table XT. — Results of modulation, ai tin time <>f sowing, nith Pythiwn 
deba/ryanum on Firms banlcsiana in different sun-inn densities in pots of 

(HI tori 1 1 red SOU. 



[The percent age of" Damping-off," columns 4 and 
:w i'ii iii columns 3, 5, 6, and 8 


r,are based on the numbei of seedlings 

ii. IT, , ,\ ,,n i he number ol seed 


the percentages 




Num- 
ber 

of 

pol s 
in 

cx- 
prri- 

11 HI 1 1 . 


Results (per ceni i. 


I lensil y of spoil sowing. 


Experiment . r >*. 


Experiment 59. 




Emerged . 


Damn- 
ing-oil. 


Sur- 
vival. 


Emerged. 


Damp- 
in "-1,11 

7 


Sur- 
vival. 


1 


2 


3 


4 


5 


6 


8 




10 

5 

5 

5 


15 
8 
1 

6 


43 
G5 
100 

33 


10 
3 


4 


2G 
8 
11 

17 


13 
91 
34 

37 


•a 


Double 


l 


Triple 

Regular, l>ut in additional seeds near 


7 
11 







MOISTURE AND TEMPERATURE FACTORS. 

The relation of damping-off to moisture and temperature are sub- 
jects less easily studied. In 11)07 and 1908 Mr. W. H. Mast, then 
supervisor of the Nebraska National Forest, conducted daily counts 
of the number of seedlings damped-off and compared these records 
with temperature and rainfall records. The writer in 1909 repeated 
his work, maintaining parallel records of damping-off, air and soil 
temperatures, soil moisture, atmospheric humidity, wind movements, 
and evaporation. The 1909 records of damping-off, temperature, 
soil moisture, and evaporation appear in figure 20. The damped-off 
seedlings were counted and removed in the morning and evening, the 
day period thus being in most cases 10 to 11 hours and the night 
period 13 to 14 hours. Because the period was not always the same 
length, the data are reduced to a per hour basis. Air temperature 
was recorded by a sheltered thermograph 3 feet above the soil sur- 
face. The evaporation graph represents the mean loss per hour 
from two porous cup atometers of the writer's own design, in which 
the rather long and slender Chamberlain filter bougie was used and 
supported in a horizontal position just above the soil surface so as 
to be under as nearly as possible the same atmospheric conditions as 
the seedlings. The two bougies were placed at right angles to each 
other in order to eliminate as far as possible the effect of change of 
wind direction on their mean loss. While the rain-correction mount- 
ing had not at that time come into use, the error due to rain absorp- 
tion appeared negligible; atometers filled shortly before rainfall 
were read immediately after without any gain being found in the 
water in the reservoir. The psychrograph and wind-movement rec- 
ords are not presented, as the evaporation valucsarc more easily inter- 
preted. Soil moisture was periodically determined in the soil of the 



76 



BULLETIN f)34, U. S. DEPARTMENT OF AGRICULTURE. 




(±H30d3c/) -0 -yflOHt/ld-O-D 

3dnj.QI0mi09 3df)lVd3dH31. NO/J.b>6/Odl//!3 



'■!) 

<3 o* G ci G ^ 

(j. N30 H3dJ dDOH if 3d AJO 
-OSdW&O 59NI1033S 



DAMPTNG-OFF IN FOREST NURSERTES. 77 

plats on which the seedling counts were conducted, each determina- 
tion representing two, and in some cases four, points. The deter- 
minations for the upper one- fourth inch of soil, made more frequently 
than for lower levels, are connected in figure 20 by a dotted line, 
which gives some idea of the amount of moisture in the surface soil 
during the periods be! ween determinations. The determinations were 
too infrequent to permit anything more than an estimate of the 
moisture conditions between determinations, but the writer, having 
before him the records of the times and amounts of rainfall and 
artificial watering as well as the evaporation and soil-moisture de- 
terminations, is in a better position to make such an estimate than 
the reader. The dotted line which gives this estimate should not be 
depended on to show what the percentage of moisture was at any one 
time, but is believed reasonably reliable as showing whether in gen- 
eral the soil was wet or dry. In interpreting the soil-moisture rec- 
ords, it should be kept in mind that the soil was very sandy, the 
wilting coefficient of composite samples from various parts of the 
nursery, as determined by the indirect method of Briggs and Shantz 
in the Laboratory of Biophysical Investigations of the Bureau of 
Plant Industry, being only 3.4 per cent. The hygroscopic moisture 
in dry air for the soil of the plats actually under consideration was 
indicated by repeated determinations for the surface soil on dry days 
to be in the neighborhood of or slightly below 2 per cent. The 
nursery is located in a region of large temperature fluctuations, where 
the air during the day is generally dry, and consequently the dew 
is heavy at night. 

The first result of interest is the difference between the damping- 
off for the day and the night periods. In the records of every 
day but two, more seedlings went down during the day period than 
during the night, the differences in most cases being large. As the 
evaporation and temperature showed similar day and night fluctua- 
tions, it is difficult to say whether temperature or moisture condi- 
tions were responsible. The other interesting result brought out by 
the graphs is the sudden drop in the general level of the damping- 
off graph following the rains of June 15, June 19-20, and July 3. 
In each of these three cases the damping-off came up again only 
after the soil moisture came down. 

The fact that in the daily fluctuations the damping-off varied 
directly with the evaporation rather than inversely is an apparent 
contradiction of the generally accepted doctrine that moisture favors 
the disease. This contradiction is, however, only apparent. Dur- 
ing the first part of the damping-off period, when the seedlings are 
still soft, the recognition of damping-off depends on the decay of 
that part of the stem just above the soil surface which allows the 
seedling to fall over. This usually takes place at this nursery as 



78 BULLETIN »34, U. S. DEPARTMENT OF AGRICULTURE. 

a result of the extension upward of Lesions which have started on 
the parts a little below the soil surface. It is supposed that such 
decay takes place most rapidly at high temperatures and that it is 
the temperature rather than the evaporation graph which the damp- 
ing-off is following in these early day and night fluctuations. Dur- 
ing the latter part of the damping-off period a dying seedling shows 
its first signs of distress in the drying up of its leaves, the stem being 
too stiff to go down until alter the infection has gone far enough 
in the roots to cut off most of the water supply. It is, of course, 
under dry conditions that such a sign of distress will be most in 
evidence. During the latter part of the damping-off period it is 
therefore altogether likely that the day and night fluctuations are 
caused, at least in part, by the higher evaporation rate which ob- 
tains during the day. This is a relation not to the rate of progress 
of the disease, but rather to the rate at tvhich the symptoms of dis- 
ease appear in plants already seriously affected. 

The drop in damping-off following the increased soil moisture of 
June 15, 19-20, and July 3 also apparently contradicts established 
doctrine. While it is ordinarily true that a wet soil is a cold soil 
and that in the rainy weather which causes wet soil the evaporation 
is usually low, it does not seem possible on inspection of the graphs 
for these items to attribute entirely the reduction of damping-off 
during these periods of wet soil either to low temperature or to low 
evaporating power of the air. Lowered soil temperature probably 
had something to do with the reduced loss following the rains. It 
is also suggested that a sudden change in moisture content may tem- 
porarily hinder a soil fungus by decreasing its air supply. In this 
samty soil the fungi can work at very considerable depths during 
dry periods. Initial lesions have been found as much as 12 inches 
below the surface. If this soil is as completely changed in its aera- 
tion qualities by wetting as the sandy soil with which Buckingham 
(19) worked, a rain might result in a rather sudden change in the level 
at which the fungus is able to operate. 

On the whole, the graphs tend to confirm the common statement 
that high temperature favors damping-off. It must, however, be 
borne in mind that in uncontrolled field plats several factors vary 
simultaneously, and it is impossible to definitely attribute any ob- 
served phenomenon to any one of them. Furthermore, it is not 
possible to say for the seedlings at different ages just how long it will 
take a factor to exert an effect on the damping-off curve. An addi- 
tional consideration is that a method of investigation which gives 
entirely reliable information on the speed with which the disease 
develops does not necessarily throw light on the conditions under 
which the greatest total amount of disease can be expected before 
the seedlings become old enough to resist attack. High temperatures, 



DAMPING-OFF IN FOREST NURSERIES. 79 

within reasonable limits, are expected to increase the speed with 
which the disease works, but these should also hasten the develop- 
ment of the host to a point at which infections are unable to cause 
death. It is the total amount of damage in the beds rather than the 
damage per unit of time which is of practical importance. For a 
number of reasons, then, the method followed in obtaining the data 
for these graphs can not give information of maximum value. While 
data of the sort mentioned are of undoubted interest and would be 
of still more value if the records had been commenced when the first 
seedlings appeared instead of a few days later, the relation of any 
specific factor to the total extent of the disease can be better deter- 
mined by comparing plats in series in which the factors are as far 
as possible controlled and varied one at a time. To vary soil moisture 
and soil temperature independently will prove somewhat difficult, 
but it can be done with the proper facilities. Some work with en- 
vironmental factors should be done under conditions of artificial in- 
oculation in the greenhouse, in which the different damping-off 
parasites can be experimented with separately, as it is obvious that 
the factors which favor the activity of one may not be favorable for 
another. 

CHEMICAL FACTORS. 

Chemical factors are presumably also important, as the soil is 
in most cases the culture medium for both the parasite and the host. 
The much greater activity of Pythium debaryanum in autoclaved 
soil than in untreated soil may be due to the larger quantity of 
soluble organic matter commonly present in autoclaved soil. Pythium 
debaryanum has been found more sensitive to unfavorable substrata 
in artificial culture than Corticium vagum and is apparently more 
dependent on soil organic matter in the nurseries than is C. vagum. 
For example, in the normal humus-containing surface sand in the 
beds at Cass Lake, Minn., both Pythium and Corticium occurred 
frequently in the damped-off seedlings, while in beds a few feet 
distant, from which enough of the surface soil had been removed to 
leave no humus, nearly all the damping-off foci contained abundant 
Corticium, and no Pythium could be found. With both fungi and, 
in addition, with two species of Fusarium (68) heavy inoculation has 
been more successful in experiments at the time of sowing than 
light inoculation. This has been thought possibly due in part to the 
larger amount of nutrient substratum added in the heavy inocula- 
tions, allowing better saprophytic development of the fungus in the 
soil. In each of the two experiments with Pythium reported in 
Table XI, a 5-pot unit was treated with corn-meal infusion and 
another with prune infusion at the time of inoculation. In both 
experiments germination was lower, damping-off after germination 
higher, and the survival less than half as great in the pots with 



80 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

infusion as in the inoculated pots not so treated. Tn the first experi- 
ment 5-pot units of unheated soil were also inoculated in the same 
way. In these also both the units which received infusions showed 
less germination and more loss after germination than the unit which 
received no infusion, though the differences were smaller than in the 
autoelaved soil. In the second experiment the light inoculation used 
failed to cause material loss in the unheated soil units, even though 
two of them were treated with the in fusion as in the previous test 
and two others received triple portions of the infusion. 

The experience in the nurseries, in which heavy applications of 
manure, and especially poorly rotted manure, in a number of cases 
have apparently resulted in increased disease, and the finding of 
Fred (43) that green manures recently plowed under favored the 
work of Corticium have already been mentioned.. The addition of 
dried blood at two nurseries in Kansas was in both cases followed 
by very much heavier loss than in the controlled plats. The only 
instances known to the writer in which the addition of organic 
matter to the soil has shown any indication of materially decreasing 
damping-ofT (with the exception, of course, of the organic disin- 
fectants) are the result reported by Gifford (46) with tankage, a 
single case in the writer's experience with bone meal, and the cases 
in which cane sugar has seemed to decrease losses somewhat (67). 
It is of some interest to note that the experience available also indi- 
cates increased disease as a result of the addition of inorganic nitrog- 
enous substances. Sodium nitrate and sodium nitrite have both 
given some indication of increasing damping-off. Ammonium sul- 
phate in six separate series has in every case resulted in decreased 
stands, though unfortunately in experiments in which the damped- 
off seedlings were not counted. Ammonium hydroxid, though ap- 
parently having some initial value as a disinfectant, as indicated by 
early damping-off losses, in a number, of cases has been followed by 
very heavy total losses. This experience is of some interest in view 
of the apparently rather general belief that plants on a soil rich 
in nitrogen are especially susceptible to disease. 

The chemical factor for which there is perhaps the most evidence 
of a relation to damping-off of conifers is acidity. The fact that 
sulphuric-acid soil treatment has been found to be one of the most 
effective means of controlling the disease, that its value is mainly 
lost if lime is later added to the soil, that soil treatment with sulphur 
in a number of cases has seemed to decrease the disease, and that 
lime alone and wood ashes have had either no effect or have appar- 
ently increased the damping-off whenever they have been tried, all 
suggest that soil acidity is not favorable to the disease. Additional 
indication of this appears in figure 12. The acidity determinations 
serving as the basis for the graph were made by Dr. L. J. Gillespie, 



DA.MJMNC (il I |\ FOREST NURSERIES. 81 

of the Bureau of Plant Industry. The estimates of the relative 
riousness of damping-off arc very approximate, based in part on 

observation only. The stations ai which damping-off is rated as 1 
are places at which it lias been reported by nurserymen or foresters 
as negligible or absent. The estimates for stations LO, 11, 11, and 15 

are based entirely on the reports of others, and for station 5 on the 
basis of counts of damped-off seedlings made by Mr. R. G. Pierce 
and Mr. Glenn (J. Ilahn. The writer personally has made the esti- 
mates or checked the estimates of the nurserymen at the other sta- 
tions. A considerable degree of correlation between the hydrogen- 
ion exponent and the amount of damping-oil' appears on the face 
of the graphs, the coefficient being 0.75 ±0.07. If the correlation is 
calculated with the H + concentration itself instead of its negative 
exponent, the coefficient, in this case itself negative, is not so high 
(— 0.58±0.11). All of the above data on acidity relation have been 
picked up incidentally in connection with other work and are merely 
suggestive. The suggestion, however, seems sufficiently strong to 
warrant further experimental work directed specifically at the rela- 
tion between soil acidity and the disease. 

The indication in the graph that damping-off is not serious in 
soils in which the hydrogen-ion exponent (P H )' ; is less than 6 is of 
particular interest, in view of the experience of Hawkins and Har- 
vev (~1) with cultures of Pythivm debaryanum on potato juice. 
The} 7 obtained good growth through a range of acidity from P H 3.4 
to 5.8, with no growth or practically none at 3.0G or 8.4. If this 
represents the acid tolerance of the fungus in the soil solution, it is 
evident that ordinarily acid soils can not be expected to remain free 
from damping-off because of inhibition of this particular fungus. 
This suggests that the apparently salutary influence of soil acidity 
in decreasing the damping-off of some of the conifers may be ex- 
erted in the direction of increasing the resistance of the host rather 
than of inhibiting the parasites. In any case, it must be kept in 
mind that as the numerous conifer hosts commonly grown in nurseries 
have many different habitat preferences and many very different 
parasites of potential importance, it is not to be expected that there 
will be found any such constant relation between any factor and the 
amount of disease as would be expected in a disease in which only a 
single parasite and a single host are involved. 

•Ph 6 is equivalent to a hydrogen-ion concentration, expressed in mols per liter, of 
lXlO- 6 or 0.000001. The higher the exponent, the smaller the hydrogen-ion concentra- 
tion. An exponent of 7 moans approximate neutrality. In dealing with this exponen- 
tial form of expression, it should be kept in mind that P H 6 means ten times and l' H 5 
one hundred times the hydrogen-iron concentration indicated by P H 7. Conversely, the 
concentration of hydroxy I ions at I'm" is one hundred times as great as at 1*h5. 

10651°— Bull. 934—21 6 



82 l.l I.I.I.'I'IX 934, U. S. DEPARTMENT OF AGRICULTURE. 

BIOLOGIC FACTORS. 

Mention has already been made of two strictly biologic factors 
which may influence the amount of damping-off. Taylor (138) and 
Rathbun (IOC) have found Fusarium not only at considerable depths 
in the soil of pine seed beds, but viable Fusarium spores without 
hyphse in the alimentary canals of earthworms and insect larvae in 
the soil, and they attribute to the migrations of these and to the 
tunnels which various animal forms make in the soil a possible im- 
portance in the distribution of damping-off Fusaria. A likely rela- 
tion between Corticiurn vagv/m epidemics in pine seed beds and the 
character of the weed flora has also been considered (GG). 

The relation between the damping-off parasites and other micro- 
organisms in the soil is also a matter of some interest. The effect of 
the microfauna of the soil on the microflora in general has been 
considered by Russell and others in a number of papers. The effect 
of soil disinfection by heat in favoring the work of artificially intro- 
duced soil-inhabiting fungous parasites, apparently a rather frequent 
phenomenon and quite evident in the inoculation experiments with 
Pyihium debcuryanum reported in the present bulletin, has been in 
other cases attributed tcr the removal of bacteria and other fungi 
which might compete with the parasites (3G, 80). Heating soil is 
known to produce physical changes and also very considerable chemi- 
cal changes both in organic and inorganic substances. These must not 
be ignored in considering the effect of previous soil heating on para- 
site activity. With a view to determining whether all the difference 
noted in the behavior of P. debaryanum in heated soil is due to the 
direct effects of the heating or in part to the elimination of com- 
peting microorganisms, an experiment was conducted in 3-inch pots 
of antoclaved soil in which 111 of them were inoculated with agar 
cultures of the Pythium at one point in each pot shortly after seed 
sowing. The seeds sown in each pot approximated 136, considerably 
more than are used on equal areas of nursery seed bed. Of these, 
fifteen 5-pot units and one 3-pot unit had been inoculated broadcast 
with rice or nutrient agar cultures of various organisms supposed to 
be saprophytic on pines. These included Phoma betae, Phoma sp., 
Chaetomium sp. (from a maple root), Rhizopus nigricans, Tricho- 
thecium roseum, Trichoderma koningi, Aspergillus spp. (including 
one with black and one with bright-colored spore heads) , Rosellinia 
sp. (from soil) , Penicillium sp., an undetermined bacterium, and three 
undetermined higher fungi. The whole 78 pots inoculated with P. 
debaryanum and saprophytes, the percentages being based on the 
total number of seeds in the case of emergence and survival and on 
the number of seedlings which appeared above ground for damping- 
off loss, as compared with those which had received the parasite only, 
gave results as follows: 



DAMPING-OFF IN FOREST NURSERIES. 83 

Pots with saprophytes: Emergence, I7.4±0.80 per cent; damplng-off, 9J per 

cent; survival, 41.0±1.23 per cent. 
Pots without saprophytes: Emergence, :'."'.7 per cent; damping-off, 14.3 per 

cciil ; survival. 20.2 per cent. 

It has been noted in the attempts to diagnose damping-off by 
planted-plate cultures that when Rhizopus appears Pythium debary- 
atiiun is not frequently obtained. It is therefore of some interest to 
note that in this case, in the two 5-pot units which received Rhizopus 
in addition, the parasite killed only 1.2 per cent and 3.3 per cent, 
respectively, of the seedlings which appeared above the soil. 

At the same time pots not inoculated with parasites were sown, 16 
other 5-pot units were inoculated with the same saprophytes as those 
used in the Pythium inoculated pots, while 25 pots were left entirely 
without inoculation. A certain amount of damping-off occurred in 
these pots also as a result of accidental infection. The results were 
as follows : 

Pols wiih saprophytes: Emergence, 17.8±0.8 per cent; damping-off, 3.9 per 

cciil : survival, 43.7±0.95 per cent. 
Pots without saprophytes: Emergence, 43.0 per cent; damping-off, r>.2 per 

cent ; survival. 38.4 per cent. 

It is of some interest to note that in these pots also the 5-pot units 
inoculated with Khizopus suffered less from damping-off than the 
average of the saprophyte-inoculated pots. 

The probable-error values given above are based on the variability 
of the emergence and survival figures of the different 5-pot units. 
No individual figures are available to serve as a basis for the deter- 
mination of the variability of the pots without saprophytes. The 
16 units which support the error determinations are, of course, not a 
sufficient number to give an entirely reliable index of variability, 
though these 16 units are respectively derived from the combination 
of a total of 78 and 80 ultimate units. The distribution of the data 
appears to be such as to justify the use of probability methods. Of 
the 64 items which went into the germination and survival calcula- 
tions, 34 showed a deviation equal to E s (probable error of a single 
unit), 9 to a deviation equal to 2E S , and only one a deviation equal 
to 3E S . 

All of the above figures are based on the results at the end of 10 
days after average emergence in the pofcs. The pots were kept on the 
benches till practically all damping-off had ceased, 36 days after 
emergence. As additional accidental infection with saprophytes 
certainly, and probably with parasites, occurred during this period, 
the results at the end of the tenth day are considered to give a better 
indication of the effect of the original inoculations. It is of some 
interest, however, to note that during the period from the tenth to 
the thirty-sixth day the difference between the pots to which sapro- 



84 BULLETIN 934, IT. s. DEPARTMENT OK AGRICULTURE. 

phytes had been added and those which received no saprophytes 
showed a slight increase, proportionally as well as in the absolute 
figures. At the end of the 3G days the survivals on all the pots were 
counted separately. The average number of seedlings per pot were 
as follows : 

Without Pythium : 

Without saprophytes, 42.8±2.3; with saprophytes, 52.1±1.1 
With Pythium: 

Without saprophytes, o0.1±2.4; with saprophytes, 4S.1±1.4. 

The difference in the survivals in favor of the pots with sapro- 
phytes in the first case is 9.3 ±2.5, three and two-thirds times its 
probable error. In the second case it is 18.0±2.8, more than six times 
its probable error. 

In general, it appears that in this experiment the inoculation of ster- 
ilized soil with saprophytes gave the seedlings some protection both 
against damping-off due to accidental infection with unidentified 
parasites and from the additional loss caused by light inoculation 
with Pythium debaryanum. The indication is, as would be expected, 
that only part of the favoring influence of heat sterilization of soil 
on introduced P. debaryanum is immediately due to the elimination 
of competition with other fungi. If a mixture of different bacteria 
and fungi had been added to each of the pots instead of but one or 
tw T o organisms to each 5-pot unit, the effect might have been more 
marked. 

It will be noted that for all the groups (fig. 18), whether with or 
without Pythium inoculation and with or without added parasites, 
the frequency polygon is asymmetrical, indicating by its shape, as 
did the frequency polygon of survivals in pots inoculated with Rheo- 
sporangium (fig. IT), that with infections which do not kill all of 
the seedlings the selection tends to be by pots rather than by seed- 
lings. In other words, in pots in which the parasites succeed in kill- 
ing any of the seedlings, they usually kill a considerable number. 
The tendency is illustrated not only by inspection of the graphs, but 
by the variability of the different groups. The greater variability 
in survivals between different pots was in both cases in the groups in 
which both the damping-off after emergence and the survival per- 
centages indicated the largest loss. The percentages of seedlings 
damped-off during the entire 3G days following emergence and the 
coefficients of variability of the survivals of the individual pots at 
the end of that time are as follows : 

Without Pythium : 

Without saprophytes, 15.5 per cent damped-off; coefficient of variability, 

39±4.2 per cent. 
With saprophytes, 11.1 per cent damped-off; coefficient of variability. 

2S±1.6 per cent. 



DAMPING-OFF IN FOREST NURSERIES. 85 

With Pythium: 

Without saprophytes, 27.5 per cenl damped-off; coefficient <>r variability, 

G7±7.S per cent. 
With saprophytes. 16.9 per rout ilainpoil-otT ; coclhcient of variability, 
39±2.4 per cent. 

This tendency has been frequently observed in experiments in 
which inoculum is applied to the soil at the time of sowing. Even 
in experiments in which a relatively small proportion of the seed- 
lings are killed, some of the pots are nearly or entirely cleaned out. 
It is taken as an indication that failure of inoculation to give results 
is often due to the inability of the fungus to maintain itself in a 
vigorous condition till the germinating seed is far enough along to 
allow easy infection. Tt may also be in part due to lack of uni- 
formity of the soil in different pots affecting virulence of parasites 
or resistance of hosts. 

In addition to this experiment on autoclaved soil, a somewhat 
similar experiment was conducted in a nursery in the Kansas sand 
hills on soil which had been treated with sulphuric acid, followed 
by lime raked into the soil. Saprophytes, for the most part the 
same strains that had been used in the experiment in the greenhouse, 
were added to 2-4 plats, each of one-half square foot, of Pinus bank- 
siana and 24 of P. pondcrosa, with 16 interspersed plats of each 
species serving as controls. The saprophytes were growing on rice, 
part of which was added to the plat with the inoculum in addition 
to the fungous mycelium. Damping-off w T as rather heavy in this soil 
from accidental infection or from parasites which survived the 
initial acid treatment, no parasites having been artificially intro- 
duced. The loss was probably due to Corticium vagum, or Fusarium 
spp. rather than to Pythium debaryanum in this case. In both pines, 
emergence was slightly better in the control plats than in those to 
which the saprophytes had been added, the difference for Pinus banh- 
siana being less than half its probable error and for P. ponderosa 
slightly more than its probable error. Damping-off for the first few 
days after emergence was somewhat less in the controls in one species, 
but higher in the saprophyte-inoculated pots in the other. The 
saprophytes therefore gave no evidence of effective competition with 
the parasites on this acid-lime treated sand. 

While the competition for water which seems to be the form of 
competition most common among green vascular plants is not likely 
to be of significance between fungi such as those which cause damping- 
off, a very little observation of the growth of mixed cultures of the 
parasites and other organisms in Petri dishes is sufficient to make 
one realize that the latter may very considerably decrease the activity 
of certain of the parasites. In nutrient agar most of the fungi and 
bacteria introduced from the soil in attempting parasite isolations. 



86 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

as well as to a less extent the paramenia, nematodes, and amoeba? 
which develop in such plates, exert a very considerable limiting in- 
fluence on the growth of most of the damping-off fungi. That they 
should also limit the growth of parasites in soil, whether by the 
production of toxic compounds, the exhaustion of food materials, or 
in other ways, seems entirely reasonable. The results in the writer's 
experiments on heated soil warrant the suggestion that further trials 
should be made of the introduction of vigorously growing bacteria 
or molds, preferably mixed cultures containing a number of different 
organisms, on seed beds which have been disinfected by some such 
method as steam or hot water, which leaves the soil in a favorable 
condition for the development of accidentally reintroduced parasites. 
If such treatment should be successful in improving the rather dis- 
appointing results with soil heating at some nurseries, it might 
easily become of practical value, as the cultivation of certain of the 
more easily grown saprophytes on a scale large enough to yield con- 
siderable quantities of bacterial or spore suspensions should be fairly 
easy and entirely practicable in an operation as intensive as that of 
raising coniferous seedlings. 

ACKNOWLEDGMENTS. 

The writer wishes to express 1 his obligations to Dr. H. A. Edson, 
of the Bureau of Plant Industry, United States Department of 
Agriculture, and to Prof. W. T. Home, of the University of Cali- 
fornia, for suggestions during the progress of this work; to Mr. Roy 
G. Pierce and Mr. Glenn G. Harm, of the Bureau of Plant Industry, 
for assistance in a number of the inoculation experiments; and to 
other members of 'the staff of the Office of Forest Pathology for 
assistance and suggestions at various times. 

SUMMARY. 

(1) Damping-ofT in nurseries is caused mainly by seedling para- 
sites which are not specialized as to host ; Pythium debaryanum and 
Corticium vagum are probably the most important of these. Damp- 
ing-off of various herbaceous hosts, including ferns, is often caused 
by specialized parasites which are limited to a particular host or 
group of hosts. Phoma betae is a rather extreme example of such 
specialization. For the conifers all the damping-off appears to be 
due to parasites of the generalized type. 

(2) Damping-off of trees, as of herbaceous plants (with the ex- 
ception of the cases caused by specialized seed-carried parasites), is 
ordinarily serious only in seed beds or cutting beds in which large 
numbers of plants are crowded together in a small space. In most of 
the forest nurseries it is a much more serious matter in conifers than 
in dicotyledonous seedlings. 



DAMPING-OFF TN FOREST NURSERI KS. 87 

(3) The most serious losses in conifers are ordinarily from the 
root-rot type of damping-off, occurring soon after the seedlings 
appear above ground and while the hypocotyls are still soft. Losses 
due to the killing of dormant or sprouting seed by parasites before 
the seedlings appear above the soil are also frequently serious, some- 
times necessarily more so than the later types, as in extreme cases 
more than half of the seed or young seedlings are destroyed in this 
way. Damping-off due to infections of parts above the soil surface 
is serious only under extremely moist atmospheric conditions. The 
late type of damping-off, in which the roots are rotted after the 
stem becomes too rigid to be easily decayed, is ordinarily less im- 
portant than the early types. Seedlings more than 2 months old are 
ordinarily able to recover from infections by the damping-off fungi. 
Even after the first month, seedlings with part of their root system 
killed often recover. 

(4) It is possible that damping-off has a certain value as a selec- 
tive agent by eliminating weak individuals in the seed-bed stage and 
allowing only the best trees to go into forest plantations. This 
value, however, is believed to be slight. Disinfectant treatments of 
seed beds, even when controlling early parasitic losses very well, 
allow a considerable percentage of disease during the last part of 
the damping-off period, often as much as occurs at the same stage of 
development in untreated beds. As it is only this late damping-off 
in which differences in individual resistance of the seedlings seem to 
be of importance in determining whether or not they 'succumb, it is 
believed that whatever selective value the disease may "have will 
appear in a larger proportion of the damping-off in the treated than 
in the untreated beds. 

(5) Of the different conifers, reports are available as to the sus- 
ceptibility of 63 species. Species which are especially susceptible 
at some nurseries may prove more resistant than the average at 
others. Pinus resinosa, which is especially subject to loss at some 
nurseries, is believed to be so because its growth at these nurseries is 
slow and its period of susceptibility is therefore especially long. 
In its early stages it does not seem especially susceptible. Repre- 
sentatives of all the commonly grown genera of the Abietoidese have 
been reported by one observer or another as decidedly susceptible. 
The reports on junipers and other members of the Cupressoidea3, on 
the other hand, have indicated a considerable amount of group re- 
sistance to damping-off. 

(6) The best control method appears to be the disinfectant treat- 
ment of the seed-bed soil before or immediately after seed is sown. 
Sulphuric acid has been found very useful for conifers, as they are 
apparently especially tolerant of acid treatment. No method has yet 
been worked out to a point at which all of its details are entirely 



88 lULl.KTlX 984, U. S. DEPARTMENT OF AGRICULTURE. 

satisfactory, though the acid treatment lias now boon in successful 
use for several years at some nurseries. At most nurseries, if the 
m in im um effective quantity of acid is used, there is no need of any 
special precautions to prevent injury to the seedlings. It is not 
expected that any single treatment can be found that can be uni- 
versally applied without change in details irrespective of differences 
in soil characters and in fungous flora. 

(7) Corticium vagum and Fusarium spp. have boon previously 
shown to be parasitic on pine seedlings. Different strains of C. 
vagum are found to vary considerably in their ability to cause damp- 
ing-off, certain strains being consistently destructive and others 
much less active in tests conducted on different species of pine and 
several years apart. The differences in activity between strains 
were greater, and apparently rather more constant from one ex- 
periment to the next, than with Peltier's strains in his carnation ex- 
periments. Comparison of the results on pine with those of Edson 
and Shapovalov on potato gives some indication that strains vigor- 
ously parasitic on one of these hosts are likely to be parasitic on the 
other also. 

(8) PytMum di baryanum, reported on many hosts and proved to 
be parasitic on few, is shown by repeated inoculation, reisolation, 
and reinoculation to be capable of causing the damping-off of seed- 
lings of pine species. The identity of the fungus causing the damp- 
ing-off of conifers with that attacking dicotyledons has been estab- 
lished by cross-inoculations as well as by morphological comparison. 
Inoculations on unheated soil are much less destructive than on 
heated soil. PytMum <1( baryanum has been obtained in culture from 
Picea engelmanni, P. sitcihensis, Tsuga merfo nsiana, Pinus banksiana, 
P. nigra austriaca, P. ponderosa] P. resinosa, and Pseudotsuga taxi- 
folia. In addition, fenugreek (TrigoneUa foenum-grm cum), cowpea 
(Vigna sp.), and rice (Oryza sativa) are reported as apparently new 
hosts among the dicotyledons. In inoculations the fungus has been 
successfully used on Pinus banksiana, P. ponderosa, P. resinosa, and 
in a preliminary experiment on Pseudotsuga taxifoMa. It had 
already been successfully used in preliminary inoculations on Picea 
canadensis by Hofmann (77). 

Differences in parasitic activity on pine are found between differ- 
ent strains of PytMum debaryanum. These differences are not as 
large and partly for this reason their constancy is not quite as con- 
clusively demonstrated as in the case of the strains of Corticium 
vagum. 

(9) Rheosponnu/ium aphanidermatus Edson, a parasite of radish 
and sugar beet, in many ways closely resembling PytMum debary- 
anum, has killed seedlings of Pinus banksiana and P. resinosa in 
certain experiments, and reisolations and reinoculations have been 



DAMPING-OFF IN FOREST NURSERIES. 89 

made. The strain available is much less destructive to the pines 
than most of the P. debaryamirn strains used, and as the fungus has 
never been isolated from coniferous seed beds it is not believed to 
be of any great importance in forest nurseries. 

(10) Phytophthora sp. from Pinus resinosa seedlings has been suc- 
cessfully used in inoculation on Pi mis resinosa and in a preliminary 
test on P. ponderosa. The strains available have been less destruc- 
tive to the pines than Pythiwm debaryanwm and the stronger strains 
of Corticium vagum. It is not common. Its relation to Phytoph- 
f/iora fagi, the European damping-off parasite of both conifers and 
dicotyledons, which has not been reported in this country, is being 
investigated. 

(11) A fungus referred to Pythiwm artotrogus, also obtained from 
damped-off Pinus resinosa, has indicated a very low degree of par- 
asitism on this host, even less than that shown by the Rheospor- 
angium and Phytophthora strains. An addition is made to the 
statements in a previous paper concerning the ability of Botrytis 
cinerea to cause damping-off in conifers. 

(12) The results of the cultural or direct examination of 742 dis- 
ease foci in seed beds of various conifers are reported. Pythium 
debaryamirn, in the plate-culture examination method, considered 
more reliable than direct examination, appeared in 51 per cent of 
the foci from untreated beds, while Corticium vagum was found in 
19 per cent. In foci in beds treated with various disinfectants, P. 
debaryanum was identified in 38 per cent of the foci and C. vagum 
in only 4 per cent. When direct microscopic examination was sub- 
stituted for isolation, C. vagum was found on a larger proportion 
of the seedlings. It was not found at all in soil which had been 
heated. The relative ease with which it appears to be controlled by 
soil disinfection is in agreement with its poor adaptation for aerial 
distribution. It was found more commonly in cases in which the 
seedlings were directly examined than when cultures were made. 
In view of the fact that at least some of the Corticium foci extend 
rapidly and include very large numbers of seedlings, it seems that 
the Corticium may be as important as P. debaryanum in causing the 
damping-off of pines. 

(13) Fusarium spp. have occurred more commonly in plate cul- 
tures than either of the above-mentioned fungi. Because little is 
known as to the parasitism of different species of this group on 
conifers, it is not possible to make any statement regarding the im- 
portance of the individual species. The evidence as a whole indi- 
cates so much importance for Pythiwm debaryanum, Corticium 
vagum, and for the Fusarium spp. considered as a group that no one 
of the three can be safely said to be more important than the others. 
None of the other fungi considered appear to be of real economic 
rank in the United States. 



90 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. 

(14) In an inoculation experiment on the roots of pines 1 .', months 
old, CorUdv/m vagwm and Pythium debdryemu/m were found able 
to cause the death of seedlings which had already developed rigid 
stems and to destroy the younger parts of the roots of seedlings which 
they were unable to kill. Indications were also obtained of similar 
but less vigorous action by Fusarium monUiforme and F. ven- 
tricosuu). 

(15) Data are given confirming the general belief that thick sow- 
ing favors the disease and indicating that soil acidity is, in general, 
unfavorable. Preliminary data on the relation of temperature and 
moisture to the disease are also presented. The parasitic activity of 
Pythi/wm debaryanum in steamed soil was in one extensive test con-, 
siderablv decreased, following the inoculation of the soil with various 
saprophytes ; this indicates both that competition of different fungi 
is a factor to be considered and that the inoculation of treated soil 
with saprophytes may sometimes prove of value in increasing the 
efficiency of heat disinfection. It is pointed out that with such a com- 
plex of parasites capable of producing identical symptoms on a num- 
ber of different hosts, no relationship between environmental factors 
and the disease can be expected to hold in all cases. 



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DAMPING-OFF IN FOREST NURSERIES. 93 

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(97) Pearson. G. W. 

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